Pharmaceutical composition for inhibiting the survival or reactivation of disseminated tumor cells

Abstract

To improve the efficiency of suppressing cancer metastasis. [Solution] A pharmaceutical composition for inhibiting the survival or reactivation of disseminated tumor cells, comprising a substance that inhibits the transcription factor TIMELESS and / or its downstream factors or downstream pathways as an active ingredient.

Description

Technical Field

[0001] The present disclosure relates to a pharmaceutical composition for suppressing the survival or reactivation of disseminated tumor cells, a pharmaceutical composition for suppressing cancer bone metastasis, an inhibitor of the transcription factor TIMELESS and / or its downstream factor or downstream pathway, a method for screening a substance that inhibits the transcription factor TIMELESS and / or its downstream factor or downstream pathway, and a method for diagnosing the presence of disseminated tumor cells.

Background Art

[0002] TIMELESS, a circadian rhythm regulator, is known to be overexpressed in cancer cells and to be involved in the enlargement of the primary tumor (Non-Patent Document 1). It has also been reported to contribute to the suppression of cell cycle arrest in cancer cells via intracellular DNA repair in a stress environment (Non-Patent Document 2), and further, there is a possibility that TIMELESS controls the stemness of cancer cells (Non-Patent Document 4).

[0003] However, when examining the respective roles of TIMELESS in each process of metastasis ((1) movement of cancer cells from the primary tumor, (2) intravascular invasion, (3) intravascular survival, (4) extravascular seeding, (5) survival of single cells or small clusters in distant metastatic foci, (6) reawakening of single cells / small clusters, (7) proliferation in distant metastatic foci), there is a report that rather, a decrease in TIMELESS expression promotes invasion from the primary tumor into blood vessels (Non-Patent Document 5). More than 90% of cancer deaths are associated with metastasis, and the circadian rhythm is deeply involved in metastasis (Non-Patent Document 6). Therefore, although analyzing the process of metastasis through the circadian rhythm is directly related to clinical treatment strategies, the role of enhanced TIMELESS expression in each process is hardly known.

[0004] Incidentally, it is believed that the most efficient stage for clinically suppressing metastasis is controlling the survival of single cells or microclusters in distant metastatic lesions and the reawakening of single cells / microclusters (Non-Patent Literature 7). This is because processes (1) to (4) have already occurred when curative treatment is performed on patients with solid tumors without distant metastases (Non-Patent Literature 8). In other words, identifying the factors that determine (5) and (6) is considered to be the most efficient metastatic treatment / prevention strategy. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Shan Zhang et al., " TIMELESS regulates sphingolipid metabolism and tumor cell growth through Sp1 / ACER2 / S1P axis in ER-positive breastcancer", Cell Death and Disease (2020) 11:892. [Non-Patent Document 2] Lauren M. Young et al., " TIMELESS Forms a Complex with PARP1 Distinctfrom Its Complex with TIPIN and Plays a Role in the DNA Damage Response", CellReports 13, 451-459, October 20, 2015. [Non-Patent Document 3] Beth K. Neilsen et al., "ERK-mediated TIMELESS expression suppresses G2 / M arrest in colon cancercells", PLoS ONE 14(1): e0209224. [Non-Patent Document 4] Limin Chi et al., " TIMELESS contributes to the progression of breast cancer through activation of MYC", Breast Cancer Research (2017)19:53. [Non-Patent Document 5] Colangelo T et al., " Loss of circadian gene Timeless inducesEMT and tumor progression in colorectal cancer via Zeb1-dependent mechanism. " Cell DeathDiffer. 2022 Aug;29(8):1552-1568. [Non-Patent Document 6] DiamantopoulouZ et al., "The metastatic spread of breast cancer acceleratesduring sleep."Nature 607,156-162 (2022). [Non-Patent Document 7] Ganesh Ket al., "Targeting metastatic cancer." Nat Med. 2021 Jan;27(1):34-44 [Non-Patent Document 8] CristSB et al., "Unchecked oxidative stress in skeletal muscle preventsoutgrowth of disseminated tumor cells." Nat Cell Biol. 2022 [Overview of the project] [Problems that the invention aims to solve]

[0006] However, conventional methods had room for improvement in metastasis suppression efficiency. Specifically, the timing of increased TIMELESS expression in the cancer metastasis process was unclear, and the metastasis suppression effect of TIMELESS inhibition was not always fully realized. Therefore, this disclosure aims to improve metastasis suppression efficiency. [Means for solving the problem]

[0007] The inventors identified the transcription factor TIMELESS as a determinant of the survival and reactivation of disseminated tumor cells. The inventors found that inhibiting the transcription factor TIMELESS while disseminated tumor cells are still alive is expected to improve the efficiency of metastasis suppression, and thus completed the present invention.

[0008] This disclosure relates, for example, to the following: [1] A pharmaceutical composition for inhibiting the survival or reactivation of disseminated tumor cells, comprising as an active ingredient a substance that inhibits the transcription factor TIMELESS and / or its downstream factors or downstream pathways. [2] The composition according to [1], wherein the disseminated tumor cells are cells that highly express both the transcription factor TIMELESS and at least one of the following factor a. Factor a: HSD17B7, ROR2, TRPS1, MGLL, TXNIP, PRKAR2B [3] The composition according to [2], wherein the disseminated tumor cells are further cells that have low expression of at least one of the following factor b. Factor b: SCD, CEBPA, CD24, TLR3, EGR1 [4] The composition according to [1], wherein the disseminated tumor cells are cells that highly express the transcription factor TIMELESS and low express at least one of the following factors b. Factor b: SCD, CEBPA, CD24, TLR3, EGR1 [5] The composition according to [4], wherein the disseminated tumor cells are further cells that highly express at least one of the following factor a. Factor a: HSD17B7, ROR2, TRPS1, MGLL, TXNIP, PRKAR2B [6] The composition according to any one of [1] to [5], wherein the substance is shRNA. [7] The composition according to [6], wherein the shRNA comprises the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. [8] The composition according to any one of [1] to [7], wherein the downstream factor or the downstream pathway is at least one selected from the group consisting of Sp1 transcription factor, mitochondrial FAS factor, and sphingolipid metabolic pathway. [9] A composition according to any one of [1] to [8] wherein the substance inhibits the transcription factor TIMELESS and / or its downstream factors or downstream pathways in at least one selected from the group consisting of bone, bone marrow, lung, brain, spinal cord, liver, kidney, ureter, bladder, lymph nodes, uterus and female adnexa, breast, male reproductive organs, muscle, subcutaneous tissue, and blood. [9'] A composition according to any one of [1] to [8] for use on at least one selected from the group consisting of bone, bone marrow, lung, brain, spinal cord, liver, kidney, ureter, bladder, lymph nodes, uterus and female adnexa, breast, male reproductive organs, muscle, subcutaneous tissue, and blood.

[10] A pharmaceutical composition for suppressing cancer bone metastasis, comprising as an active ingredient a substance that inhibits the transcription factor TIMELESS and / or its downstream factors or downstream pathways in at least one selected from the group consisting of bone, bone marrow, lung, brain, spinal cord, liver, kidney, ureter, bladder, lymph nodes, uterus and female adnexa, breast, male reproductive organs, muscle, subcutaneous tissue, and blood. [10'] A pharmaceutical composition for inhibiting cancer bone metastasis, comprising as an active ingredient a substance that inhibits the transcription factor TIMELESS and / or its downstream factors or downstream pathways, for use on at least one selected from the group consisting of bone, bone marrow, lung, brain, spinal cord, liver, kidney, ureter, bladder, lymph nodes, uterus and female adnexa, breast, male reproductive organs, muscle, subcutaneous tissue, and blood.

[11] An inhibitor of the transcription factor TIMELESS and / or its downstream factors or downstream pathway, comprising a nucleic acid consisting of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

[12] A method for screening substances that inhibit the transcription factor TIMELESS and / or its downstream factors or downstream pathways, (a) Step of collecting disseminated tumor cells from a subject in the remission period of cancer, (b) Step of contacting the disseminated tumor cells with a test compound, (c) Step of measuring the expression levels of Sox2, Sox9 and / or CD44 in the disseminated tumor cells, (d) Step of comparing each of the expression levels with the expression levels of Sox2, Sox9 and / or CD44 in control disseminated tumor cells not contacted with the test compound, and (e) Step of selecting, as the substance, the test compound in which the expression level in the disseminated tumor cells contacted with the test compound is lower than the expression level in the control disseminated tumor cells. A method comprising the above steps.

[13] Measuring the expression levels of at least one of the transcription factor TIMELESS and the following factor a in a sample, and comparing at least one of the transcription factor TIMELESS and the factor a with a control level. When at least one of the transcription factor TIMELESS and the factor a is highly expressed compared to the control level, a method for diagnosing the presence of disseminated tumor cells. Factor a: HSD17B7, ROR2, TRPS1, MGLL, TXNIP, PRKAR2B

[14] The method according to

[13] , for diagnosing the presence of disseminated tumor cells when at least one of the following factor b is lowly expressed compared to the control level. Factor b: SCD, CEBPA, CD24, TLR3, EGR1

[15] Measuring the expression levels of at least one of the transcription factor TIMELESS and the following factor b in a sample, and comparing at least one of the transcription factor TIMELESS and the factor b with a control level. When the transcription factor TIMELESS is highly expressed compared to the control level and at least one of the factor b is lowly expressed compared to the control level, a method for diagnosing the presence of disseminated tumor cells. Factor b: SCD, CEBPA, CD24, TLR3, EGR1

[16] The method according to

[15] , for diagnosing the presence of disseminated tumor cells, when at least one of the following factors a is highly expressed as compared to the level of the control. Factor a: HSD17B7, ROR2, TRPS1, MGLL, TXNIP, PRKAR2B

[17] A method for suppressing the survival or reactivation of disseminated tumor cells, comprising administering a substance that inhibits the transcription factor TIMELESS and / or its downstream factor or downstream pathway to a subject that needs it.

[18] A substance that inhibits the transcription factor TIMELESS and / or its downstream factor or downstream pathway, for use in suppressing the survival or reactivation of disseminated tumor cells.

[19] Use of a substance that inhibits the transcription factor TIMELESS and / or its downstream factor or downstream pathway for the manufacture of a pharmaceutical composition for suppressing the survival or reactivation of disseminated tumor cells.

[20] The method according to any one of

[17] to

[19] , the substance for use, or the use, wherein the disseminated tumor cells are cells in which at least one of the transcription factor TIMELESS and the following factor a is highly expressed. Factor a: HSD17B7, ROR2, TRPS1, MGLL, TXNIP, PRKAR2B

[21] The method according to

[20] , the substance for use, or the use, wherein the disseminated tumor cells are cells in which at least one of the following factor b is lowly expressed. Factor b: SCD, CEBPA, CD24, TLR3, EGR1

[22] The method according to any one of

[17] to

[19] , the substance for use, or the use, wherein the disseminated tumor cells are cells in which the transcription factor TIMELESS is highly expressed and at least one of the following factor b is lowly expressed. Factor b: SCD, CEBPA, CD24, TLR3, EGR1

[23] The method according to

[22] , the use of a substance that inhibits the transcription factor TIMELESS and / or its downstream factors or downstream pathways, wherein the disseminated tumor cells are further cells that highly express at least one of the following factor a. Factor a: HSD17B7, ROR2, TRPS1, MGLL, TXNIP, PRKAR2B

[24] The method described in any of

[17] to

[23] , the use of the transcription factor TIMELESS and / or its downstream factors or downstream pathways, or the use thereof, wherein the substance is shRNA.

[25] The method described in

[24] , the use of the transcription factor TIMELESS and / or its downstream factors or downstream pathways, or the use thereof, wherein the shRNA comprises the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[26] The method according to any one of

[17] to

[25] , a substance that inhibits the transcription factor TIMELESS and / or its downstream factor or downstream pathway for use, wherein the downstream factor or downstream pathway is at least one selected from the group consisting of Sp1 transcription factor, mitochondrial FAS factor, and sphingolipid metabolic pathway, or use thereof.

[27] The method according to any one of

[17] to

[26] , a substance that inhibits the transcription factor TIMELESS and / or its downstream factors or downstream pathways in at least one selected from the group consisting of bone, bone marrow, lung, brain, spinal cord, liver, kidney, ureter, bladder, lymph nodes, uterus and female adnexa, breast, male reproductive organs, muscle, subcutaneous tissue, and blood, or a use thereof.

[28] A method for suppressing cancer bone metastasis, comprising administering a substance that inhibits the transcription factor TIMELESS and / or its downstream factors or downstream pathways in at least one selected from the group consisting of bone, bone marrow, lungs, brain, spinal cord, liver, kidneys, ureters, bladder, lymph nodes, uterus and female adnexa, breasts, male reproductive organs, muscles, subcutaneous tissue, and blood, to a subject in need thereof.

[29] Substances for use in suppressing bone metastases of cancer that inhibit the transcription factor TIMELESS and / or its downstream factors or downstream pathways in at least one selected from the group consisting of bone, bone marrow, lung, brain, spinal cord, liver, kidney, ureter, bladder, lymph nodes, uterus and female adnexa, breast, male reproductive organs, muscle, subcutaneous tissue, and blood.

[30] Use of a substance that inhibits the transcription factor TIMELESS and / or its downstream factors or downstream pathways in at least one selected from the group consisting of bone, bone marrow, lung, brain, spinal cord, liver, kidney, ureter, bladder, lymph nodes, uterus and female adnexa, breast, male reproductive organs, muscle, subcutaneous tissue, and blood, for the manufacture of a pharmaceutical composition for suppressing cancer bone metastasis.

[31] A method for inhibiting the transcription factor TIMELESS and / or its downstream factors or downstream pathways, comprising administering a nucleic acid consisting of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 to a subject in need thereof.

[32] Nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 for use in the inhibition of the transcription factor TIMELESS and / or its downstream factors or downstream pathways.

[33] Use of nucleic acids comprising the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 for the manufacture of the transcription factor TIMELESS and / or its downstream factors or inhibitors of downstream pathways.

[34] A method for detecting disseminated tumor cells, comprising measuring the expression levels of the transcription factor TIMELESS and at least one of the following factor a in a sample, comparing the levels of the transcription factor TIMELESS and at least one of the factor a with control levels, and determining that disseminated tumor cells are present if both the transcription factor TIMELESS and at least one of the factor a are expressed at levels higher than the control levels. Factor a: HSD17B7, ROR2, TRPS1, MGLL, TXNIP, PRKAR2B

[35] The method according to

[34] , further comprising confirming that at least one of the following factors b is expressed at a lower level compared to the control level. Factor b: SCD, CEBPA, CD24, TLR3, EGR1

[36] A method for detecting disseminated tumor cells, comprising measuring the expression levels of the transcription factor TIMELESS and at least one of the following factors b in a sample, comparing the levels of the transcription factor TIMELESS and at least one of the factors b with those of a control, and determining that disseminated tumor cells are present if the transcription factor TIMELESS is expressed at a higher level than the control and at least one of the factors b is expressed at a lower level than the control. Factor b: SCD, CEBPA, CD24, TLR3, EGR1

[37] The method according to

[36] , further comprising confirming that at least one of the following factors a is highly expressed compared to the control level. Factor a: HSD17B7, ROR2, TRPS1, MGLL, TXNIP, PRKAR2B [Effects of the Invention]

[0009] According to this disclosure, the efficiency of metastasis suppression can be improved. According to this disclosure, cancer recurrence can also be suppressed. According to one embodiment of this disclosure, an inhibitor of the transcription factor TIMELESS and / or its downstream factors or downstream pathways can be provided. According to this inhibitor, the survival or reactivation of disseminated tumor cells can be suppressed. According to one embodiment of this disclosure, a pharmaceutical composition for suppressing the survival or reactivation of disseminated tumor cells can be provided. By suppressing the survival or reactivation of disseminated tumor cells, subsequent macroscopic metastasis can be suppressed more efficiently. According to one embodiment of this disclosure, a pharmaceutical composition for suppressing cancer bone metastasis can be provided.

[0010] According to one embodiment of this disclosure, a method can be provided for screening substances that inhibit the transcription factor TIMELESS and / or its downstream factors or downstream pathways.

[0011] According to one embodiment of this disclosure, a method for diagnosing the presence of disseminated tumor cells can be provided. This allows for the decision to apply a substance that inhibits the transcription factor TIMELESS and / or its downstream factors or pathways to subjects with disseminated tumor cells, thereby improving the efficiency of treatment. Unnecessary treatment can be avoided for subjects without disseminated tumor cells. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1(a) shows the change in tumor cell count in a mouse model in which breast cancer tumor cells were transplanted into the tail artery. Figure 1(b) shows the change in tumor cell count in a mouse model in which bladder cancer tumor cells were transplanted into the tail artery. [Figure 2] Figure 2 shows transmitted light and fluorescence images of disseminated tumor cells. [Figure 3] Figure 3(a) shows the expression level of the stem cell gene Sox2. Figure 3(b) shows the results of gene set enrichment analysis related to fatty acid metabolism. [Figure 4] Figure 4 is a flowchart of the gene screening used to decide to target TIMELESS. [Figure 5] Figure 5 shows the expression levels of Sox2 (Figure 5(a)), Sox9 (Figure 5(b)), CD44 (Figure 5(c)), OXSM (Figure 5(d)), HSD17B7 (Figure 5(e)), and HSD17B8 (Figure 5(f)) in tumor cells treated with a pharmaceutical composition containing a TIMELESS inhibitor. [Figure 6] Figure 6 shows the survival rate of metastatic initiation cells in tumor cells (Figure 6(a): breast cancer, Figure 6(b): breast cancer, Figure 6(c): prostate cancer, or Figure 6(d): bladder cancer) treated with a pharmaceutical composition containing a TIMELESS inhibitor. [Figure 7] Figure 7 shows the results of macroscopic metastasis observations of breast cancer tumor cells treated with a pharmaceutical composition containing a TIMELESS inhibitor. [Figure 8]Figure 8 shows the results of observations regarding visceral metastasis in breast cancer tumor cells treated with a pharmaceutical composition containing a TIMELESS inhibitor. [Figure 9] Figure 9 is a schematic diagram showing some of the downstream signals of TIMELESS. [Figure 10] Figure 10 shows the effect of downstream signaling inhibitors on bone tumor formation potential of TIMELESS. [Figure 11] Figure 11 shows the effect of TIMELESS downstream signaling inhibitors on body weight changes in mice. [Figure 12] Figure 12(a) shows the overall survival rate for each level of TIMELESS protein expression in primary tumor resection specimens. Figure 12(b) shows the overall survival rate for each level of TIMELESS protein expression in primary tumor resection specimens. [Figure 13] Figure 13 shows the expression pattern of TIMELESS protein in a primary tumor resection specimen. [Figure 14] Figure 14(a) shows the cell proliferation curves of control and TIMELESS knockdown cell lines in the MCF7 (breast cancer) cell line. Figure 14(b) shows the cell proliferation curves of control and TIMELESS knockdown cell lines in the UM-UC-3 (bladder cancer) cell line. Figure 14(c) shows the cell proliferation curves of control and TIMELESS knockdown cell lines in the PC-3 (prostate cancer) cell line. [Figure 15] Figure 15(a) shows the oxygen consumption rate in control and TIMELESS knockdown cell lines. Figure 15(b) shows the basal respiratory volume in control and TIMELESS knockdown cell lines. Figure 15(c) shows the oxygen consumption rate in control and MPA-08 treated cells. Figure 15(d) shows the basal respiratory volume in control and MPA-08 treated cells. Figure 15(e) shows the cell count in cells treated with MPA-08 and cisplatin, respectively. [Modes for carrying out the invention]

[0013] The following describes the forms for implementing this disclosure, but this disclosure is not limited to the following embodiments.

[0014] The first aspect of this disclosure relates to a pharmaceutical composition for inhibiting the survival or reactivation of disseminated tumor cells, comprising a substance that inhibits the transcription factor TIMELESS and / or its downstream factors or pathways as an active ingredient.

[0015] One embodiment of the second aspect of this disclosure is a pharmaceutical composition for inhibiting bone metastasis of cancer, comprising as an active ingredient a substance that inhibits the transcription factor TIMELESS and / or its downstream factors or downstream pathways in at least one selected from the group consisting of bone, bone marrow, lung, brain, spinal cord, liver, kidney, ureter, bladder, lymph nodes, uterus and female adnexa, breast, male reproductive organs, muscle, subcutaneous tissue, and blood. Another embodiment of the second aspect of this disclosure is a pharmaceutical composition for inhibiting bone metastasis of cancer, comprising as an active ingredient a substance that inhibits the transcription factor TIMELESS and / or its downstream factors or downstream pathways, for use in at least one selected from the group consisting of bone, bone marrow, lung, brain, spinal cord, liver, kidney, ureter, bladder, lymph nodes, uterus and female adnexa, breast, male reproductive organs, muscle, subcutaneous tissue, and blood.

[0016] A third aspect of this disclosure is an inhibitor of the transcription factor TIMELESS and / or its downstream factors or downstream pathways, comprising a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

[0017] In this specification, "containing as an active ingredient" means "containing as an active ingredient," and also means "containing in an effective amount" of a substance. In this specification, "effective amount" means the amount of drug or medicine that elicits a biological or medical response in a tissue, system, animal or human being as required by a researcher or physician.

[0018] In this specification, "substances that inhibit the transcription factor TIMELESS and / or its downstream factors or downstream pathways" are also referred to as "inhibitors." In this specification, inhibitors may be substances that inhibit the transcription factor TIMELESS and / or its downstream factors or downstream pathways in at least one selected from the group consisting of bone, bone marrow, lungs, brain, spinal cord, liver, kidneys, ureters, bladder, lymph nodes, uterus and female adnexa, breasts, male reproductive organs, muscles, subcutaneous tissue, and blood. Inhibitors in this specification may be small molecule compounds that specifically suppress the expression of the transcription factor TIMELESS and / or its downstream factors. Specifically suppressing the expression of the transcription factor TIMELESS and / or its downstream factors means that the expression of the transcription factor TIMELESS and / or its downstream factors is strongly suppressed, but the expression of other mRNAs and other proteins is not suppressed, or is only weakly suppressed. The inhibitors described herein may be nucleic acids that suppress the gene expression of the transcription factor TIMELESS or its downstream factors, such as antisense oligonucleotides, siRNAs, shRNAs, miRNAs, and ribozymes. The inhibitors described herein may be nucleic acids consisting of at least one nucleotide sequence selected from the group consisting of the nucleotide sequence shown in SEQ ID NO: 1 and a nucleotide sequence having 85% or more, 90% or more, 95% or more, or 98% or more sequence identity therewith, and the nucleotide sequence shown in SEQ ID NO: 2 and a nucleotide sequence having 85% or more, 90% or more, 95% or more, or 98% or more sequence identity therewith. The inhibitors described herein are preferably nucleic acids consisting of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0019] The antisense oligonucleotide against the gene for the transcription factor TIMELESS or its downstream factor may be the entire sequence or a partial sequence of the complementary strand of the mRNA encoding the transcription factor TIMELESS or its downstream factor. The antisense oligonucleotide may be DNA, RNA, or a DNA / RNA chimera. The number of bases in the antisense oligonucleotide may be 10-50 bases, 20-100 bases, 100-1000 bases, 100-10000 bases, or 100-100000 bases.

[0020] The number of bases in an siRNA may be 10-50, 15-30, or 20-25. siRNA may have an additional base at its 5' or 3' end. The length of this additional base is usually about 2-4 bases, resulting in a total length of 19 bases or more for the siRNA. This additional base may be DNA or RNA; however, using DNA may improve the stability of the nucleic acid.

[0021] The inhibitor in this specification is preferably shRNA. More preferably, the inhibitor in this specification is shRNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. The number of nucleotides of the shRNA may be 10 to 50 nucleotides, 15 to 30 nucleotides, or 20 to 25 nucleotides.

[0022] The number of base pairs in a miRNA may be 10 to 50 base pairs, 20 to 100 base pairs, or 100 to 100,000 base pairs.

[0023] siRNA, shRNA, and miRNA can be designed based on the cDNA sequence information (target sequence) of the target gene.

[0024] Examples of ribozymes include self-splicing RNAs found in infectious RNAs such as viroids and virusoids, and hammerhead and hairpin types are known. The number of bases in a ribozyme may be 10-50 bases, 20-200 bases, 100-1000 bases, or 100-5000 bases.

[0025] In this specification, the downstream factors or pathways of the transcription factor TIMELESS may be at least one selected from the group consisting of the Sp1 transcription factor, the mitochondrial FAS factor, and the sphingolipid metabolic pathway.

[0026] The pharmaceutical compositions and inhibitors relating to this disclosure may contain the inhibitor at a concentration of 0.01% by mass or more, 0.1% by mass or more, or 1.0% by mass or more, based on the total mass of the pharmaceutical composition. The pharmaceutical compositions and inhibitors relating to this disclosure may contain the inhibitor at a concentration of 30% by mass or less, 15% by mass or less, or 5.0% by mass or less, based on the total mass of the pharmaceutical composition. The pharmaceutical compositions and inhibitors relating to this disclosure may contain the inhibitor at a concentration of 0.1 mg / ml or more, 1.0 mg / ml or more, or 10 mg / ml or more. The pharmaceutical compositions and inhibitors relating to this disclosure may contain the inhibitor at a concentration of 300 mg / ml or less, 150 mg / ml or less, or 50 mg / ml or less.

[0027] The dosage of the pharmaceutical composition and inhibitors relating to this disclosure may be 0.1 to 5000 mg / day, 1.0 to 500 mg / day, or 10 to 50 mg / day in terms of the mass of the inhibitor when administered to a human (adult, weighing 60 kg). These amounts may be administered at once or in multiple doses. The pharmaceutical composition and inhibitors relating to this disclosure may be administered daily.

[0028] The pharmaceutical compositions and inhibitors relating to this disclosure may be administered to mammals such as humans, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cattle, horses, goats, and monkeys.

[0029] The pharmaceutical compositions and inhibitors relating to this disclosure may be administered after or concurrently with treatment for cancer, such as surgery, chemotherapy, or radiotherapy. The pharmaceutical compositions and inhibitors relating to this disclosure may be administered after the primary tumor of cancer has been removed. That is, the pharmaceutical compositions and inhibitors relating to this disclosure may be administered to subjects from whom the primary tumor of cancer has been removed. The subjects to whom the pharmaceutical compositions and inhibitors relating to this disclosure may be subjects in remission of cancer. In other words, the pharmaceutical compositions and inhibitors relating to this disclosure may be administered during the remission phase of cancer. The pharmaceutical compositions and inhibitors relating to this disclosure may be pharmaceutical compositions and inhibitors for maintaining remission. Cancer remission is a state in which the symptoms of cancer are temporarily reduced or disappear. Remission may be partial remission or complete remission. The pharmaceutical compositions and inhibitors relating to this disclosure may be administered to subjects diagnosed with a high risk of cancer metastasis or recurrence. The pharmaceutical compositions and inhibitors relating to this disclosure may be administered to subjects with a history of cancer metastasis or recurrence. The pharmaceutical compositions and inhibitors relating to this disclosure, when administered to these subjects or at these times, can further suppress cancer metastasis or recurrence in those subjects. Because the pharmaceutical compositions and inhibitors relating to this disclosure can suppress cancer metastasis and recurrence, they can also be considered therapeutic agents for the complete cure of cancer.

[0030] In this specification, cancer may be at least one selected from the group consisting of breast cancer, bladder cancer, and prostate cancer. Breast cancer may be at least one selected from the group consisting of estrogen receptor-positive breast cancer and triple-negative breast cancer.

[0031] The pharmaceutical compositions and inhibitors relating to this disclosure may be applied to at least one selected from the group consisting of bone, bone marrow, lung, brain, spinal cord, liver, kidney, ureter, bladder, lymph nodes, uterus and female adnexa, breast, male reproductive organs, muscle, subcutaneous tissue, and blood. The pharmaceutical compositions or inhibitors relating to this disclosure can be applied to at least one selected from the group consisting of bone, bone marrow, lung, brain, spinal cord, liver, kidney, ureter, bladder, lymph nodes, uterus and female adnexa, breast, male reproductive organs, muscle, subcutaneous tissue, and blood by systemic or topical administration in accordance with conventional methods.

[0032] After some cells acquire invasiveness in the primary tumor, they may migrate into the stroma and blood vessels and survive as circulating tumor cells. When circulating tumor cells are disseminated outside the blood vessels, they remain dormant and survive as disseminated tumor cells. Subsequent reactivation of disseminated tumor cells can lead to cancer metastasis and recurrence. Disseminated tumor cells can usually be defined as cancer cells that have migrated to other sites via the bloodstream or lymphatic system away from the primary tumor, but this does not rule out their presence in or around the primary tumor. Reactivation of disseminated tumor cells in a site far from the primary tumor can lead to cancer metastasis, and reactivation of disseminated tumor cells in or around the primary tumor can lead to cancer recurrence. Disseminated tumor cells may include cells that initiate metastasis. Cells that initiate metastasis may be cells that have the ability to survive for a medium to long period as single cells or small tumor cell clusters in sites other than the primary tumor and then reactivate. Disseminated tumor cells can also be described as cells in the (metastatic) latency phase.

[0033] In this specification, disseminated tumor cells may be cells that highly express the transcription factor TIMELESS, cells that highly express both the transcription factor TIMELESS and at least one of the factors a below, cells that highly express the transcription factor TIMELESS and at least one of the factors b below, or cells that highly express both the transcription factor TIMELESS and at least one of the factors a below and at least one of the factors b below. "Highly expressed" means that the transcription factor TIMELESS, factor a, and factor b are expressed at a level higher than the expression levels of these factors in control cells, such as primary tumor cells, circulating tumor cells in the blood, cells extracted from these cells and subjected to some kind of artificial manipulation, or tumor-derived tissue obtained from the blood. For example, if the expression level of these factors in control cells is 120% or higher, or 150% or higher, with the expression level in control cells being 100%, then these factors can be said to be "highly expressed." "Low expression" of these factors means that their expression levels are lower compared to those of control primary tumors, circulating tumor cells in the blood, cells extracted from them and subjected to some kind of artificial manipulation, or tumor-derived tissue from the blood. For example, if the expression level of these factors in the control is set at 100%, then these factors can be said to be "lowly expressed" if it is 80% or less, 90% or less, or 70% or less. Factor a: HSD17B7, ROR2, TRPS1, MGLL, TXNIP, PRKAR2B Factor b: SCD, CEBPA, CD24, TLR3, EGR1

[0034] The expression levels of the transcription factors TIMELESS, factor a, and factor b may be expressed as protein levels or mRNA levels. Expression levels can be measured by known methods such as Western blotting, ELISA, Northern blotting, and RT-PCR.

[0035] The pharmaceutical compositions and inhibitors relating to this disclosure may be administered orally or parenterally (e.g., intravenously, subcutaneously, topically, rectally, percutaneously, intrathecally, or nasally). Dosage forms for oral administration include, for example, tablets, capsules, pills, granules, powders, solutions, and suspensions. Dosage forms for parenteral administration include, for example, aqueous solutions for injection and oily solutions for injection. These formulations may be prepared using conventionally known techniques. The pharmaceutical compositions and inhibitors relating to this disclosure may also be prepared in a form in which the gene has been introduced into cells. In this case, the cells into which the gene has been introduced may be incorporated into phosphate-buffered saline, Ringer's solution, intracellular composition solutions for injection, etc.

[0036] The pharmaceutical compositions and inhibitors relating to this disclosure may contain pharmaceutically acceptable additives as necessary. In the case of solid preparations, pharmaceutically acceptable excipients such as starch, lactose, refined sucrose, glucose, crystalline cellulose, carboxycellulose, carboxymethylcellulose, carboxyethylcellulose, calcium phosphate, magnesium stearate, gum arabic, etc., may be added during formulation, and lubricants, binders, disintegrants, coatings, colorants, etc. may be added if necessary. In the case of liquid preparations, stabilizers, solubilizers, suspending agents, emulsifiers, buffers, preservatives, etc. may be added.

[0037] The fourth aspect of this disclosure is a method for screening substances that inhibit the transcription factor TIMELESS and / or its downstream factors or downstream pathways, (a) A process of collecting disseminated tumor cells from a subject in remission of cancer, (b) A step of contacting the disseminated tumor cells with the test compound, (c) A step of measuring the expression levels of Sox2, Sox9 and / or CD44 in the disseminated tumor cells, (d) A step of comparing the expression levels with those of Sox2, Sox9, and / or CD44 in control disseminated tumor cells that have not been exposed to the test compound, and (e) A method comprising the step of selecting a test compound as the substance such that the expression level in the disseminated tumor cells contacted with the test compound is lower than the expression level in the control disseminated tumor cells.

[0038] The "subjects" may be mammals such as humans, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, and monkeys. These animals (other than humans) may be experimental model animals or pets.

[0039] (a) The step may involve collecting potentially disseminated tumor cells from a subject in cancer remission and confirming in vitro that the collected cells are disseminated tumor cells. Disseminated tumor cells can be confirmed if the collected cells highly express the transcription factor TIMELESS or factor a, or lowly express factor b. Gene expression levels can be measured by methods known to those skilled in the art. Such methods include, for example, reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, Northern blotting, RNA sequencing, microarrays, Western blotting, radioimmunoassay, and ELISA.

[0040] (b) step may be a step of contacting the cells collected in step (a) with the test compound or test substance. Step (b) can be carried out by any method that allows the test compound or test substance to come into contact with the cells. For example, when using cultured cells, a method of adding the test compound or test substance to the culture medium may be used. For example, when bringing the test compound or test substance into contact with target cells in a living organism, systemic administration such as oral administration, intravenous administration, intraperitoneal administration, or local administration to a target organ or target tissue may be used.

[0041] Examples of test compounds or substances that can be used include nucleic acids, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, cell culture supernatants, plant extracts, mammalian tissue extracts, plasma, etc. The test compounds or substances may be novel compounds or substances, or known compounds or substances. These test compounds or substances may form pharmaceutically acceptable salts.

[0042] (c) The measurement of expression levels in step (c) may be either the measurement of protein quantity or the measurement of mRNA quantity. In either case, the expression level can be measured by a standard method. For example, when measuring protein quantity, Western blotting or ELISA can be used. When measuring mRNA quantity, Northern blotting or RT-PCR can be used.

[0043] (d) The step may be to compare the expression levels of Sox2, Sox9, and / or CD44 (hereinafter also referred to as the "target expression level") in disseminated tumor cells that have been contacted with the test compound or test substance with the expression levels of Sox2, Sox9, and / or CD44 (hereinafter also referred to as the "control expression level") in control disseminated tumor cells that have not been contacted with either the test compound or the test substance. In step (d), the expression levels of factors other than Sox2, Sox9, and CD44 (other factors) may also be compared between disseminated tumor cells and control disseminated tumor cells. WNT5A is an example of other factors.

[0044] (e) The step may be a step of selecting a test compound or test substance as an inhibitor, such that the target expression level is 80% or less, 90% or less, or 70% or less of the control expression level.

[0045] One embodiment of the fifth aspect of this disclosure is a method for diagnosing the presence of disseminated tumor cells if both the transcription factor TIMELESS and at least one of the following factor a are expressed at a higher level than in the control, comprising measuring the expression levels of the transcription factor TIMELESS and at least one of the following factor a in a sample, and comparing the expression levels of the transcription factor TIMELESS and at least one of the following factor a are expressed at a higher level than in the control. Factor a: HSD17B7, ROR2, TRPS1, MGLL, TXNIP, PRKAR2B

[0046] One embodiment of the fifth aspect of this disclosure may further include measuring the expression level of at least one of the following factor b in a sample, and further including comparing the expression level of at least one of the following factor b in a control with the expression level of at least one of the following factor b in a control. If the expression level of at least one of the following factor b in a sample is lower than the expression level in a control, it can be more reliably determined that disseminated tumor cells are present. Factor b: SCD, CEBPA, CD24, TLR3, EGR1

[0047] Another embodiment of the fifth aspect of this disclosure is a method for diagnosing the presence of disseminated tumor cells, comprising measuring the expression levels of the transcription factor TIMELESS and at least one of the following factors b in a sample, and comparing the expression levels of the transcription factor TIMELESS and at least one of the following factors b in a control, wherein the transcription factor TIMELESS is expressed at a higher level than in the control and at least one of the following factors b is expressed at a lower level than in the control. Factor b: SCD, CEBPA, CD24, TLR3, EGR1

[0048] Another embodiment of the fifth aspect of this disclosure may further include measuring the expression level of at least one of the following factor a in a sample, and further including comparing the expression level of at least one of the following factor a in a control with the expression level of at least one of the following factor a in a control. If the expression level of at least one of the following factor a in a sample is higher than the expression level in a control, it can be more reliably determined that disseminated tumor cells are present. Factor a: HSD17B7, ROR2, TRPS1, MGLL, TXNIP, PRKAR2B

[0049] The sample may be derived from the subject and may be at least one selected from the group consisting of bone specimens, bone marrow specimens, lung specimens, brain specimens, spinal cord specimens, liver specimens, kidney specimens, ureteral specimens, bladder specimens, lymph node specimens, uterine and female adnexal specimens, breast specimens, male reproductive organ specimens, muscle specimens, subcutaneous tissue specimens, and blood specimens.

[0050] In the fifth aspect of this disclosure, "highly expressed" transcription factors TIMELESS, factor a, and factor b means that their expression levels are higher than those of control primary tumors, circulating tumor cells in the blood, cells extracted from them and subjected to some artificial manipulation, or tumor-derived tissue from the blood. For example, if the expression level of these factors in the control is set at 100%, then these factors can be said to be "highly expressed" if it is 120% or higher or 150% or higher. "Lowly expressed" transcription factors mean that their expression levels are lower than those of control primary tumors, circulating tumor cells in the blood, cells extracted from them and subjected to some artificial manipulation, or tumor-derived tissue from the blood. For example, if the expression level of these factors in the control is set at 80% or lower, 90% or lower, or 70% or lower, then these factors can be said to be "lowly expressed" if it is 80% or lower, 90% or lower, or 70% or lower, compared to 100% of the expression level of these factors in the control.

[0051] The measurement of expression levels refers to the measurement of the expression level of a protein or mRNA. Expression levels can be measured by known methods such as Western blotting, ELISA, Northern blotting, and RT-PCR.

[0052] A method for diagnosing the presence of disseminated tumor cells can also be called a companion diagnostic method. Generally, a companion diagnostic is the prediction of the effectiveness of a drug for the treatment, prevention, or maintenance of remission of a disease without the actual administration of the drug. The transcription factor TIMELESS, factor a, and factor b can also be called companion diagnostic biomarkers in this specification. A companion diagnostic biomarker means a biomarker for companion diagnostics.

[0053] Methods for diagnosing the presence of disseminated tumor cells can also be described as methods to assist in diagnosing the presence of disseminated tumor cells, methods for determining the presence of disseminated tumor cells, methods for diagnosing cancer recurrence, methods to assist in diagnosing cancer recurrence, or methods for determining cancer recurrence. These diagnostic methods, diagnostic assisting methods, and determination methods can be performed in vitro. [Examples]

[0054] The present disclosure will be described in more detail below using examples, but the present disclosure is not limited to the following examples.

[0055] <Creation of a mouse model of bone metastasis with disseminated tumor cells> Female nude mice Balb / cA Jcl nu / nu (Clea Japan, Inc, Tokyo, Japan), 6-7 weeks old, were anesthetized with 1.75% isoflurane, and tumor cells were transplanted into the tail artery. 1x10 6Individual MCF cells and UM-UC-3 cells (n = 3 each) were suspended in 100 μL of PBS and injected using a 1 mL disposable syringe with a 30G needle. The IVIS Spectrum imaging system (Caliper Life-Science, Hopkinton, MA, USA) was used to confirm the engraftment and proliferation of tumor cells. After the mice were anesthetized全身 with 1.75% isoflurane after transplantation of tumor cells, VivoGlo Luciferin in vivo Grade (Promega) was administered intraperitoneally, and imaging was performed 15 minutes later under the conditions of an exposure time of 1 minute, Medium, and Stage C. The results are shown in Figure 1.

[0056] <Confirmation of metastatic initiating cells (MICs) in frozen sections> Fourteen days after transplantation of tumor cells into the tail vein, the mice were euthanized, the knee joints were excised, placed in a container together with an embedding agent (SCEM, Leica Microsystems, Section-Lab), and cryo-embedded using hexane. The obtained blocks were trimmed and sectioned to a thickness of 8 μm using an adhesive film (Cryofilm, Leica Microsystems, Section-Lab). Tumor cells were confirmed by checking the expression of red fluorescent protein with a fluorescence microscope IX-73 (OLYMPUS, Tokyo, Japan). The results are shown in Figure 2. For the tumor cells extracted from the knee joints, the expression status of the stem cell factor Sox2 was examined, and gene set enrichment analysis was also performed. The results are shown in Figure 3.

[0057] <Culture of MICs strains> [[ID=,12]]Fourteen days after transplantation of tumor cells into the tail vein, the mice were euthanized, the knee joints were excised, placed in 3 mL of PBS supplemented with 2% FBS and 1 mM EDTA, and the tissue was mashed using a sharp blade. The well-crushed tissue was incubated in a 37°C incubator for 40 minutes and then filtered using a cell strainer (70 μm). After centrifuging the cells (1200 rpm, 7 minutes), they were cultured in DMEM containing 0.5 mg / mL zeocin to establish MICs strains.

[0058] <Screening Using Comprehensive Gene Analysis Information of Clinical Specimens> Transcriptome data of 253 cases of estrogen receptor-positive (ER+) breast cancer published in public data were collected. Transcriptome data of estrogen receptor-negative (ER-) breast cancer specimens in the same dataset were also collected. The relative expression levels of each gene in ER+ breast cancer were analyzed with the expression level of each gene in ER- breast cancer as a control. As a result, genes specifically expressed in ER+ breast cancer were identified. To further narrow down the identified genes (candidate genes), univariate analysis was performed on the candidate genes extracted from the mouse model. Univariate analysis identified whether each gene affected the prognosis such as survival rate and recurrence rate. Multivariate analysis was performed using factors with significantly poor prognosis. Multivariate analysis identified genes strongly related to the prognosis. Among them, factors related to fatty acid synthase were extracted, and one of them was the transcription factor TIMELESS. The flowchart used for screening is shown in Figure 4.

[0059] <Application of a Pharmaceutical Composition Containing an Inhibitory Component of TIMELESS to Tumor Cells> A pharmaceutical composition containing an inhibitor of TIMELESS was applied to tumor cells. As inhibitors of TIMELESS, sh-1 and sh-2, which are hairpin-type RNAs (shRNAs), were used. The sequence of sh-1 is 5’-GCTAGAGATTGTCTCCCTTAT-3’: SEQ ID NO: 1. The sequence of sh-2 is 5’-GTGTTTGGTATTAAACATGTA-3’: SEQ ID NO: 2. A lentiviral vector pLKO1 (plasmid #8453 addgene) was used for introducing these shRNAs into tumor cells. As tumor cells, MCF7 cells, MDA-MB-231 cells, PC-3 cells and UM-UC-3 cells were used. After introducing sh-1 and sh-2 into these tumor cells, drug selection was performed with 0.2 - 0.5 μg / ml of puromycin (Invitrogen) to obtain resistant colonies. In the cells after drug selection, the expression levels of Sox2, Sox9, CD44, OXSM, HSD17B7 and HSD17B8 were examined. The results are shown in Figure 5.

[0060] <Evaluation of intramedullary survival, reactivation, and secondary visceral seeding of tumor cells to which a pharmaceutical composition containing a TIMELSS inhibitor was applied> For in vivo experiments evaluating factors regulating the survival and reactivation of intramedullary tumor cells, female nude mice Balb / cA Jcl nu / nu (Clea Japan) at 7 - 8 weeks of age were used. The nude mice were transplanted with the parental strains of MCF cells (1.0×10 6 cells), MDA - MB - 231 cells (5.0×10 5 cells), and UM - UC - 3 cells (1.0×10 6 cells), as well as tumor cells to which a pharmaceutical composition containing a TIMELSS inhibitor was applied, via the caudal artery, and the intramedullary survival ability and reactivation ability were evaluated over time using an IVIS Spectrum imaging system (Caliper Life - Science). On the other hand, for the caudal artery transplantation of PC - 3 cells (1.0×10 6 cells), female NOD / ShiJic - scid nude mice (Clea Japan) at 5 - 6 weeks of age were used and evaluated in the same manner. The survival rate of tumor cells to which a pharmaceutical composition containing a TIMELSS inhibitor was applied during the metastatic latency period is shown in Fig. 6. Macroscopic metastases were observed in mice 42 days after caudal artery transplantation of MCF cells (1.0×10 6 cells) or tumor cells to which a pharmaceutical composition containing a TIMELSS inhibitor was applied. The results are shown in Fig. 7. Thereafter, the tumor volumes in the brain and lungs were also examined. The results are shown in Fig. 8.

[0061] <Verification of the effect of suppressing in vivo intramedullary survival and macroscopic metastatic growth by a TIMELSS downstream pathway inhibitor> Female NOD / ShiJic - scid nude mice (Clea Japan, Inc.) at 8 - 9 weeks of age were given 2.5×10 5One MDA-MB-231 cell was transplanted into the tail artery. Two days after transplantation, mice in which tumor cells had engrafted in the knee were divided into three groups. From the third day after transplantation, the inhibitor group was administered MPA-08 (100 mg / kg), a sphingosine kinase 1 / 2 inhibitor, six times a week, while the control group was administered vehicle (70% [vol / vol] PEG 400) six times a week. The remaining group was administered cisplatin (5 mg / kg) twice a week. A schematic diagram showing that MPA-08 inhibits sphingosine kinase 1 / 2 is shown in Figure 9. The changes in tumor cell volume after transplantation are shown in Figure 10. The percentage increase in body weight at 28 days post-transplantation relative to body weight before tail artery transplantation is shown in Figure 11.

[0062] <Immunohistochemical staining of human estrogen-positive breast cancer surgical specimens> From 2007 to 2017, among patients with stage III estrogen-positive breast cancer who underwent surgical resection at Hokkaido Cancer Center and Saitama Prefectural Cancer Center, we excluded cases where sufficient clinical information was unavailable or where evaluation was difficult due to the condition of the specimen. We then observed the expression patterns of TIMELESS in specimens from a total of 213 patients. For observation, immunohistochemical staining was performed using standard methods. Formalin-fixed, paraffin-embedded tissue was sectioned, and then hematoxylin-eosin staining and immunohistochemical staining with anti-TIMELESS antibody (ab109512) were performed to observe the expression patterns. Expression intensity was quantified using Fiji and classified into two groups: high expression and low expression. Figure 12 shows the non-bone metastasis rate and overall survival rate after resection of the primary breast cancer lesion. Figure 13 shows the results of immunohistochemical staining.

[0063] <Result> A mouse model of bone metastasis was created in which tumor cells injected into the tail artery were in a metastatic latency phase or in a reactivated state (Figure 1). Tumor cells 14 days after injection were rich in both growth inhibitory factors and reactivating factors, and were therefore considered to be MICs. As shown in Figure 2, tumor cells at this stage were almost entirely isolated. In fact, in the cell population extracted from tumor cells at this stage, as shown in Figure 3(a), proliferative capacity decreased and the expression of stem cell factors increased. In addition, as shown in Figure 3(b), tumor cells at this stage had an enriched set of genes involved in fatty acid synthesis. When a screening model for extracting poor prognostic factors was used for tumor cells at this stage, TIMELESS, which is both a circadian rhythm factor and a transcription factor, was extracted as the factor with the lowest p-value (Figure 4). In MICs that undergo dormancy, the fact that circadian rhythm factors control prognosis is consistent with the dormancy mechanism, and the results that this factor is a survival determinant were consistent statistically, genetically, and biologically.

[0064] In tumor cells treated with a pharmaceutical composition containing a TIMELESS inhibitor, TIMELESS regulated two other factors (HSD17B7 and OXSM) in addition to stem cell factors (Sox2, Sox9, and CD44) (Figure 5). HSD17B7 and OXSM are factors that regulate fatty acid synthesis. As shown in Figure 6, in MCF7 and UM-UC-3, the signal intensity of Sh-TIM1 and Sh-TIM2 was significantly suppressed compared to the Sh-control at 14 days post-injection. This confirmed that tumor cells treated with a pharmaceutical composition containing a TIMELESS inhibitor had a reduced survival rate during the metastatic latency period. In addition, it was demonstrated that macroscopic metastasis was suppressed when tumor cells treated with a pharmaceutical composition containing a TIMELESS inhibitor were transplanted into the tail artery (Figure 7). A reduction in secondary visceral tumors due to reduced bone tumor formation was also confirmed (Figure 8). We applied MPA-08, a research-stage inhibitor, to the sphingolipid pathway, one of the downstream signaling pathways of TIMELESS (Figure 9). As a result, we found that the identified drug significantly suppressed in vivo tumorigenesis compared to cisplatin, one of the most widely used drugs in clinical practice (Figure 10), and that its effect on mouse body weight changes was unclear (Figure 11). Regarding resected specimens of primary breast cancer, it was shown that high levels of TIMELESS protein expression were associated with a higher risk of bone metastasis and a lower overall survival rate (Figure 12). In other words, it was found that the prognosis differed depending on the expression level of TIMELESS. From this, we demonstrated that TIMELESS is a useful factor for predicting prognosis at the time of primary tumor resection. Furthermore, we confirmed that TIMELESS protein is expressed heterogeneously in resected specimens of primary breast cancer (Figure 13).

[0065] This embodiment demonstrates that the pharmaceutical composition or inhibitor of this disclosure can suppress the survival or reactivation of disseminated tumor cells. Therefore, the pharmaceutical composition or inhibitor of this disclosure can efficiently suppress cancer metastasis or recurrence.

[0066] <Reference Experiment Example 1: Verification of the role of TIMELESS in proliferating cell lines> The changes in cell count over 8 days after general culture were compared between control and TIMELESS knockdown cell lines in MCF7 (breast cancer), UM-UC-3 (bladder cancer), and PC-3 (prostate cancer). Specifically, TIMELESS knockdown cell lines (ShTIM-1) and control cell lines (Sh-control) were prepared for each of the MCF7, UM-UC-3, and PC-3 cell lines. Next, each cell line was seeded in a 96-well plate. After seeding, the cells were cultured in an incubator at 37°C and 5% CO2. Cell counts were measured on days 0, 2, 4, 6, and 8 after seeding using a commercially available cell proliferation assay kit. The results are shown in Figure 14.

[0067] <Result> After 8 days of culture, there were no significant differences in cell number among the cell lines (Figure 14). This confirms that TIMELESS knockdown does not affect cell proliferation in the MCF7, UM-UC-3, and PC-3 cell lines. This suggests that a reduction in TIMELESS does not directly affect the proliferative capacity of cancer cells in the proliferative phase.

[0068] <Reference Experiment Example 2: Verification of the role of TIMELESS in oxidative phosphorylation> Timeless knockdown cell lines (ShTIM-2) and control cell groups (Sh-control) were prepared for the MCF7 cell line. Oxidative phosphorylation was measured in each cell group using an XF analyzer (Seahorse XF Analyzer). Each cell line was seeded on an XF plate and cultured for 24 hours, after which the cellular oxygen consumption rate (OCR) was measured. Based on the obtained OCR values, the basal respiratory capacity was calculated. MCF7 cell lines cultured in normal medium were administered MPA-08 (30 μM). After 48 hours of culture, OCR was measured and basal respiratory capacity was calculated as above, and the number of cells was counted. MCF7 cell lines cultured in normal medium were administered cisplatin (30 μM). After 48 hours of culture, the number of cells was counted. The results are shown in Figure 15.

[0069] <Result> Cisplatin at the same concentration as MPA-08 (30 μM) significantly inhibited cell proliferation more effectively than MPA-08 (Figure 15). Knockdown of TIMELESS and treatment with MPA-08 significantly suppressed oxidative phosphorylation. Furthermore, MPA-08 showed a potentially smaller inhibitory effect on cell proliferation compared to cisplatin. This suggests that TIMELESS plays an important role in regulating oxidative phosphorylation.

[0070] These results reveal that while a decrease in TIMELESS does not directly affect the proliferative capacity of cancer cells in the proliferative phase, TIMELESS plays a role in regulating oxidative phosphorylation.

Claims

1. A pharmaceutical composition for inhibiting the survival or reactivation of disseminated tumor cells, comprising a substance that inhibits the transcription factor TIMELESS and / or its downstream factors or pathways as an active ingredient.

2. The composition according to claim 1, wherein the disseminated tumor cells are cells that highly express both the transcription factor TIMELESS and at least one of the following factor a. Factor a: HSD17B7, ROR2, TRPS1, MGLL, TXNIP, PRKAR2B

3. The composition according to claim 2, wherein the disseminated tumor cells are further cells that have low expression of at least one of the following factor b. Factor b: SCD, CEBPA, CD24, TLR3, EGR1

4. The composition according to claim 1, wherein the disseminated tumor cells are cells that highly express the transcription factor TIMELESS and lowly express at least one of the following factors b. Factor b: SCD, CEBPA, CD24, TLR3, EGR1

5. The composition according to claim 4, wherein the disseminated tumor cells are further cells that highly express at least one of the following factor a. Factor a: HSD17B7, ROR2, TRPS1, MGLL, TXNIP, PRKAR2B

6. The composition according to claim 1, wherein the substance is shRNA.

7. The composition according to claim 6, wherein the shRNA consists of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

8. The composition according to claim 1, wherein the downstream factor or the downstream pathway is at least one selected from the group consisting of Sp1 transcription factor, mitochondrial FAS factor, and sphingolipid metabolic pathway.

9. The composition according to claim 1, for use on at least one selected from the group consisting of bone, bone marrow, lung, brain, spinal cord, liver, kidney, ureter, bladder, lymph nodes, uterus and female adnexa, breast, male reproductive organs, muscle, subcutaneous tissue, and blood.

10. A pharmaceutical composition for inhibiting cancer bone metastasis, comprising a substance that inhibits the transcription factor TIMELESS and / or its downstream factors or downstream pathways, for use on at least one selected from the group consisting of bone, bone marrow, lung, brain, spinal cord, liver, kidney, ureter, bladder, lymph nodes, uterus and female adnexa, breast, male reproductive organs, muscle, subcutaneous tissue, and blood, as an active ingredient.

11. An inhibitor of the transcription factor TIMELESS and / or its downstream factors or downstream pathways, comprising a nucleic acid consisting of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

12. A method for screening substances that inhibit the transcription factor TIMELESS and / or its downstream factors or downstream pathways, (a) A process of collecting disseminated tumor cells from a subject in remission of cancer, (b) A step of contacting the disseminated tumor cells with the test compound, (c) A step of measuring the expression levels of Sox2, Sox9 and / or CD44 in the disseminated tumor cells, (d) A step of comparing the expression levels with those of Sox2, Sox9, and / or CD44 in control disseminated tumor cells that have not been exposed to the test compound, and (e) A step of selecting the test compound as the substance such that the expression level in the disseminated tumor cells that have been contacted with the test compound is lower than the expression level in the control disseminated tumor cells. A method that includes this.

13. A method for diagnosing the presence of disseminated tumor cells, comprising measuring the expression levels of the transcription factor TIMELESS and at least one of the following factor a in a sample, and comparing the expression levels of the transcription factor TIMELESS and at least one of the factor a with control levels, wherein both the transcription factor TIMELESS and at least one of the factor a are expressed at a higher level than the control level. Factor a: HSD17B7, ROR2, TRPS1, MGLL, TXNIP, PRKAR2B

14. The method according to claim 13, for diagnosing the presence of disseminated tumor cells if at least one of the following factors b is expressed at a lower level compared to the control level. Factor b: SCD, CEBPA, CD24, TLR3, EGR1

15. A method for diagnosing the presence of disseminated tumor cells, comprising measuring the expression levels of the transcription factor TIMELESS and at least one of the following factor b in a sample, and comparing the expression levels of the transcription factor TIMELESS and at least one of the factor b with those of a control, wherein the transcription factor TIMELESS is expressed at a higher level than the control and at least one of the factor b is expressed at a lower level than the control. Factor b: SCD, CEBPA, CD24, TLR3, EGR1

16. The method according to claim 15, for diagnosing the presence of disseminated tumor cells if at least one of the following factors a is expressed at a higher level compared to the control level. Factor a: HSD17B7, ROR2, TRPS1, MGLL, TXNIP, PRKAR2B

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