WO2016064034A1

WO2016064034A1 - Composition for inhibiting cancer stem cell growth and cancer metastasis containing tspyl5 expression or activation inhibitor

Info

Publication number: WO2016064034A1
Application number: PCT/KR2015/000856
Authority: WO
Inventors: In-Gyu Kim; Seo Yoen KIM; Soo Im Choi; Jae Ha Lee; Kug Chan Kim; Byung Chul Shin
Original assignee: Korea Atomic Energy Research Institute
Priority date: 2014-10-23
Filing date: 2015-01-27
Publication date: 2016-04-28
Also published as: KR20160047735A

Abstract

The present invention relates to a pharmaceutical composition for inhibiting cancer metastasis comprising the TSPYL5 (testis-specific protein, Y-encoded-like 5) expression or activity inhibitor as an active ingredient. When the non-small cell lung cancer cell line A549 was irradiated with the fractionated radiation, the expressions of TSPLY5 (testis-specific protein, Y-encoded-like 5) and the cancer stem cell markers ALDH1 and CD44 were significantly increased. The inhibition of TSPYL5 expression or activity resulted in the suppression of the expressions of the cancer stem cell markers ALDH1 and CD44, by which the functions of EMT, the major pathway of cancer stem cell growth and metastasis, were significantly reduced and accordingly the growth of malignant cancer cells was inhibited. Therefore, the TSPYL5 expression or activity inhibitor can be effectively used as an active ingredient of a composition for inhibiting the cancer stem cell growth and cancer metastasis in various cancers.

Description

[DESCRIPTION]

[invention Title]

COMPOSITION FOR INHIBITING CANCER STEM CELL GROWTH AND CANCER METASTASIS CONTAINING TSPYL5 EXPRESSION OR ACTIVATION INHIBITOR

[Technical Field]

The present invention relates to a composition for inhibiting cancer stem cell growth and metastasis comprising the TSPYL5 (testis-specific protein, Y-encoded- like 5) expression or activity inhibitor as an active ingredient, and a method for screening an anti-cancer metastasis material candidate using the said TSPYL5 and the cancer stem cell marker ALDH.

[Background Art]

According to the increase in the number of cancer patients every year, the treatment cases using anticancer agents and radiotherapy have been increased as well. Approximately 30 ~ 50% of cancer patients are treated with radiotherapy. Radiotherapy, which is used to reduce the size of a tumor by using radiation before surgical operation and used to eliminate malignant cancer cells survived from the surgical operation, generally indicates the fractionated radiotherapy characterized by the repeat of comparatively low dose of radiation in order to reduce side effects of killing normal cells by high dose of radiation. The effectiveness of such radiotherapy differs from the characteristics of cancer, patient, and other anticancer agents co- treated, etc. In general, the cancer that is treated by radiotherapy is exemplified by uterine cancer, lung cancer, pharynx cancer, brain cancer, breast cancer, colorectal cancer, larynx cancer, and head/neck cancer. Even though these types of cancers are major targets of radiotherapy, the response to radiotherapy of these cancers is often not as good as expected or even though the early treatment seems successful, there are many cases of re-occur and displaying ill-progress.

Lung cancer takes the number one position in death rate of cancer. That is, the cure rate of this disease is the lowest. Even if the early therapeutic response was satisfactory, the 5 year survival rate is less than 15% (Jemal A, Siegel R, Cancer statistics. CA. Cancer. J. Clin. 56(2006)106-130]) . In this invention, lung cancer indicates the cancer originated from the lung cancer cell, which is largely classified into small cell lung cancer and non-small cell lung cancer. Small cell lung cancer takes approximately 15% of the total lung cancer, which is mainly developed in the inside of the bronchus or near the bronchus and displays fast progress, compared with non- small cell lung cancer. Non- small cell lung cancer is again divided into three types: which are adenocarcinoma, squamous cell carcinoma (SCC) , and large cell carcinoma. Among them, adenocarcinoma takes approximately 40% of the total non- small cell lung cancer and is generally developed in the peripheral bronchus. Squamous cell carcinoma takes about 25% of the total non-small cell lung cancer and is generally started in the center of the bronchus. Large cell carcinoma is presumed to be originated from neuroendocrine cells and might be observed together with other types of non-small cell lung cancers. As explained above, small cell or non- small cell lung cancer is very different in genetic characteristics, histological characteristics, immunological phenotype, and required clinical treatment way (Travis WD. Pathology of lung cancer. Clin. Chest . Med. 32 (2011) 669692; Spira A, Ettinger DS . Multidisciplinary management of lung cancer. N Engl. J. Med. 350 (2004) 379-392) . The recent argument saying cancer stem cells are the major reason of cancer recurrence after radiotherapy is persuasive. Cancer stem cells are the specific cell group that are^' able to be self-renewed constantly and have various pulprint potency like stem cells. So, even with a small number of cancer stem cells, a tumor can be formed in a test animal. The cancer stem cells show a surprisingly- strong resistance against radiotherapy and chemotherapy, which are necessary treatment methods for cancer treatment (B.M. Boman, M.S. icha, Cancer stem cells: a step toward the cure, J. Clin. Oncol. 26(2008) 2795-2799). The cancer stem cells were first identified in acute myeloid leukemia and have been recently identified in general solid tumors including breast cancer as well, suggesting that solid tumors also contain the stem cells (D. Bonnet, J.E. Dick, Human acute myeloid leukemia is organized as hierarchy that originates from a primitive hematopoietic cell. Nat. Med. 3(1997)730- 737; M. Al-Hajj, M.F. Clarke, Self-renewal and tumor stem cells. Oncogene. 23(2003)7274-7284). In particular, CD133 (prominin-1 or AC133) , the transmembrane protein, was used as a marker for the recognition and separation of cancer stem cells in brain tumor cases. When more or less than 100 CD133+ cells were transplanted in NOD-mouse, a tumor was formed (S.K. Singh, C. Hawkins, I.D. Clarke, et al, Identification of human brain tumor initiating cells. Nature. 432(2004) 396-401). Another transmembrane protein CD44 (hyaluronate receptor or P- glycoprotein 1) is also used as a cancer stem cell marker. For example, the cells separated by CD44 in cooperation with other markers caused xenograft tumor growth in breast cancer cases (M. Al-Hajj, M.S. Wicha, A. Bentino-Hernandez et al, prospective identification of tumorigenic breast cancer cells. Proc . Natl. Acad. Sci . USA. 100 (2003) 3983- 3988) . Along with the transmembrane proteins CD133 and CD44, aldehyde dehydrogenasel (ALDH1) is another promising cancer stem cell marker.

ALDH1 is a detoxifying enzyme that oxidizes intracellular aldehyde, which displays a strong resistance against alkylating agent or oxidative stress (M. Magni, S. Shammah, R. Schiro. et al, Induction of cyclophosphamide- resistance by aldehyde-dehydrogenase gene transfer. Blood. 87(1996) 1097-1103; N.A. Sophos , V. Vasiliou, Aldehyde dehydrogenase gene superfamily: the 2002 update, Chem. Biol Interact. 143-144(2003)5-22). ALDH converts retinol (Vitamin A) into retinoic acid that is the most active form of retinoids playing an important role in the treatment and prevention of cancer, indicating that ALDH also plays a certain role in cell growth and proliferation. It is generally known that ALDH contains 19 genes, which are classified as ALDH 1, II, and III classes. Among them, only ALDH1 has a retinal dehydrogenase activity that can convert retinal into retinoic acid. Along with ALDH1, ALDH1A3 (retinaldehyde dehydrogenase 3) shows the most efficient activity (A. Sima, M. Parisotto, S. Mader, P.V. Bhat, kinetic characterization of recombinant mouse retinal dehydrogenase type 3 and 4 for retinal substrates. Biochem. Biophys. Acta. 1790(2009) 1660-1664). Therefore, the ALDH1 activity can be effectively used for the separation of a subpopulation of cancer stem cells in various cancer cell lines including lung cancer cells (J. Feng, Q. Qiu, K Abha et al, Aldehyde dehydrogenase 1 is a tumor stem cell- associated marker in lung cancer, Mol . Cancer. Res. 7(2009) 330-338) . To avoid recurrence of cancer and metastasis and further to eliminate cancer completely, it is important to go over the limit of current cancer treatment methods that only attack the cancer cells and to target cancer stem cells showing the characteristics of stem cells so as to develop a novel anticancer agent to destroy cancer stem cells .

TSPYL5 gene is a member of the testis-specific protein Y-encoded- like (TSPY-L) family, which is located on chromosome 8q22. This gene is mainly expressed in breast cancer and is one of 70 genes suspected to play an important role in the development of breast_, cancer (van't Veer, L. J. et al . , Nature. 415:530-536, 2002). This gene is also considered as one of 10 genes which are potential classification genes to distinguish head/neck cancer (Head and neck Squamous Cell Carcinoma) from lung squamous cell carcinoma (Kim, T.Y. et al . , Clin. Cancer Res. 13(10) :2905- 2915, 2007) . However, the cellular physiological functions of TSPYL5 gene have not been disclosed, yet.

The recent cancer treatment depends mainly on surgical operation, chemotherapy, and radiotherapy. Except surgical operation, chemotherapy and radiotherapy require various drugs and radiation, but their application is limited to how far human can hold up. Because of such limitation of application of chemotherapy and radiotherapy, even though it is proved to be very excellent in treatment effect in animal test, the clinical effect of such treatment in human becomes weaker than expected. This is also a kind of side effect attributed to the generation of anticancer resistant cancer cells or cytotoxicity accompanied with each therapy. For example, cisplatin is the most effective anticancer agent, which is the most useful pharmaceutical agent among 30 kinds of anticancer drugs used clinically in these days. Cisplatin has been known to show anticancer effect on testis cancer, ovarian cancer, lung cancer, head/neck cancer, bladder cancer, stomach cancer, uterine cervical cancer, etc. (Teni Boulikas, Oncology Reports, 10:1663-1682, 2003). However, the cancers having resistance against cisplatin have been confirmed, according to recent reports. Since it is very difficult to treat such resistant cancer cells and there is a high risk of recurrence of such cancer after the treatment finished, studies are actively going on the combined therapy of cisplatin and other chemical materials or co-treatment of cisplatin together with the regulation of intracellular protein expression (Tito Fojo, Oncogene, 22: 7512-7523, 2003). Radiotherapy is an anticancer method to treat various human cancers by irradiating a proper amount of radiation on the affected area. This radiotherapy reduces the hemopoietic function in human and inhibits the immune system, so that the efficiency in cancer treatment by this radiotherapy decreases. Further, the repeated irradiation builds the resistance of cancer cells, and thus radiotherapy is also limited in its treatment effect.

In the course of study to overcome the above problems, the present inventors irradiated fractionated radiation on the non-small cell lung cancer cell line A549. As a result, the inventors confirmed that the expressions of TSPYL5 and the cancer stem cell markers ALDH1 and CD44 were all increased by the fractionated irradiation. The present inventors also confirmed that the inhibition of TSPYL5 expression or activation resulted in the suppression of EMT functions, the major pathway of cancer cell growth and metastasis. That is, the present inventors confirmed that the TSPYL5 expression or activation inhibitor can be effectively used as an active ingredient of a pharmaceutical composition for inhibiting cancer metastasis, leading to the completion of the invention.

[Disclosure]

[Technical Problem]

It is an object of the present invention to provide a pharmaceutical composition for preventing and inhibiting cancer metastasis comprising the TSPYL5 (testis-specific protein, Y-encoded-like 5) expression or activity inhibitor as an active ingredient. [Technical Solution]

To achieve the above object, the present invention provides a pharmaceutical composition for preventing and inhibiting cancer metastasis comprising the TSPYL5 (testis- specific protein, Y-encoded-like 5) expression or activity inhibitor as an active ingredient.

The present invention also provides a method for preventing cancer metastasis containing the step of administering a pharmaceutically effective dose of the TSPYL5 expression or activity inhibitor to a subject having cancer. The present invention further provides a method for inhibiting cancer metastasis containing the step of administering a pharmaceutically effective dose of the TSPYL5 expression or activity inhibitor to a subject having cancer.

The present invention also provides a use of the TSPYL5 expression or activity inhibitor for a pharmaceutical composition for preventing and inhibiting cancer metastasis.

The present invention also provides a kit for inhibiting cancer stem cell growth comprising the TSPYL5 expression or activity inhibitor.

The present invention also provides a use of the kit for inhibiting cancer stem cell growth comprising the TSPYL5 expression or activity inhibitor.

The present invention also provides a method for inhibiting cancer stem cell growth containing the step of treating the TSPYL5 expression or activity inhibitor to cancer cells.

The present invention also provides a method for screening a cancer metastasis inhibitor candidate comprising the following steps:

1) preparing the cell line expressing TSPYL5;

2) treating the sample material to the cell line of step 1) ; 3) measuring the expression or activity of ALDH in the cell line; and

4) selecting the sample material that could inhibit the expression or activity of ALDH, compared with the expression or activity thereof in the control not-treated with the sample material .

In addition, the present invention provides a method for screening a cancer stem cell growth inhibitor candidate comprising the following steps:

1) preparing the cell line expressing TSPYL5 ;

2) treating the sample material to the cell line of step 1) ;

3) measuring the expression or activity of ALDH in the cell line; and

4) selecting the sample material that could reduce the expression or- activity of ALDH, compared with the expression or activity thereof in the control not-treated with the sample material . [Advantageous Effect]

When the non-small cell lung cancer cell line A549 was irradiated with the fractionated radiation, the expressions of TSPLY5 (testis-specific protein, Y-encoded- like 5) and the cancer stem cell markers ALDH1 and CD44 were significantly increased. The inhibition of TSPYL5 expression or activity resulted in the suppression of the expressions of the cancer stem cell markers ALDHl and CD44, by which the functions of EMT, the major pathway of cancer stem cell growth and metastasis, were significantly reduced and accordingly the growth of malignant cancer cells was inhibited. Therefore, the TSPYL5 expression or activity inhibitor can be effectively used as an active ingredient of a composition for inhibiting the cancer stem cell growth and cancer metastasis in various cancers.

[Description of Drawings]

The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein.- Figure 1 is a diagram illustrating the results of irradiating the lung cancer cell line with fractionated radiation:

(A) : the expressions of TSPYL5, ALDH1A1, ALDHlA3 , CD44, Sox2, 0ct4, PTEN, Jaggedl, and β-catenin after the irradiation with the fractionated radiation, confirmed by Western blotting; and

(B) : the expressions of TSPYL5 and ALDHlAl after the irradiation with the fractionated radiation ( 2 Gy x 3) , confirmed by immunofluorescence.

Figure 2 is a diagram illustrating the correlation between EMT and the fractionated irradiation in the lung cancer cell line:

(A) : the migration and invasion in the lung cancer cell line irradiated with the fractionated radiation; and (B) : the expressions of the EMT markers N-cadherin,

E-cadherin, and Snaillin the lung cancer cell line irradiated with the fractionated radiation, confirmed by Western blotting.

Figure 3 (A) illustrates the separation of ALDHl active cells and ALDHl inactive cells by using FACS after staining A549 cells with Aldefluor, Figure 3 (B) illustrates the results of colony formation assay with ALDHl (+, high) active cells displaying the lung cancer stem cell activity and ALDHl (- , low) inactive cells, Figure 3(C) illustrates the expressions of the cancer stem cell markers ALDH1A1 , ALDHl 3 , CD44, and CD133 genes in ALDHl (+, high) active cells and ALDHl (-, low) inactive cells, and Figure 3(D) illustrates the expressions of the cancer stem cell markers ALDH1A1, ALDHlA3 , CD44 , and CD133 proteins in ALDHl (+, high) active cells and ALDHl (- , low) inactive cells.

Figure 4 (A) illustrates the results of Western blotting and PCR performed to investigate the correlation of the cancer stem cell marker ALDHl and the over- expression and inhibition of TSPYL5 gene in the lung cancer cell line, Figure 4(B) illustrates the results of colony

1.3 formation assay performed to investigate the effect of TSPYL5 over-expression and inhibition on cell growth in the lung cancer cell line, Figure 4(C) illustrates the expression changes of ALDHl when TSPYL5 was over-expressed or inhibited, confirmed by FACS after staining the cells with aldeflour, and Figure 4 (D) is a diagram illustrating the results of PCR, Western blotting, and colony formation assay performed with ALDHl (+, high) active cells in which TSPYL5 was inhibited.

Figure 5 is a diagram illustrating the metastasis according to the inhibition and over-expression of TSPYL5 gene in the lung cancer cell line:

(A) and (C) illustrate the results of Western blotting performed to investigate the expressions of the EMT markers N-cadherin, E-cadherin, Twist, Vimentin, and Snaill according to the inhibition and over-expression of TSPYL5 gene in the lung cancer cell line, and (B) and (D) illustrate the invasion and migration of cancer cells mediated by the inhibition and over-expression of TSPYL5 gene in the lung cancer cell line.

Figure 6 is a diagram illustrating the cancer stem cell growth affected by the inhibition and over-expression of TSPYL5 gene in the lung cancer cell line:

(A) illustrates the sphere formation of A549 cell line wherein TSPYL5 was inhibited constantly by shRNA- TSPYL5, confirmed by Western blotting, and (B) illustrates the sphere formation of H460 cell line wherein TSPYL5 was over-expressed, confirmed by Western blotting.

Figure 7 is a diagram illustrating the expressions of TSPYL5 protein and the lung cancer stem cell markers after ALDH1 gene was inhibited or over-expressed therein in order to investigate the correlation of the lung cancer stem cell marker ALDH1 and TSPYL5 :

(A) illustrates the expressions of the cancer stem cell markers CD44, CD133, Sox2 , Oct3/4, Nanog, and β- catenin and the expression of TSPYL5 after the cancer stem cell markers ALDH1A1 and ALDH1A3 were inhibited, confirmed by Western blotting, (B) illustrates the results of colony formation assay performed to investigate the cell growth after the inhibition of ALDH1A1 and ALDH1A3 , (C) illustrates the expressions of the cancer stem cell markers CD44, CD133, Sox2, Oct3/4, Nanog, and β-catenin and the expression of TSPYL5 after the cancer stem cell markers ALDH1A1 and ALDH1A3 were over-expressed, confirmed by Western blotting, and (D) illustrates the results of colony formation assay performed to investigate the cell growth after the over-expression of ALDH1A1 and ALDH1A3.

Figure 8 is a diagram illustrating the metastasis according to the inhibition and over-expression of ALDH1 gene in the lung cancer cell line: (A) and (C) illustrate the results of Western blotting performed to investigate the expressions of the EMT markers N-cadherin, E-cadherin, Twist, Vimentin, and Snaill according to the inhibition and over-expression of ALDH1 gene in the lung cancer cell line, and (B) and (D) illustrate the invasion and migration of cancer cells mediated by the inhibition and over-expression of ALDH1 gene in the lung cancer cell line.

Figure 9 is a diagram illustrating the results of sphere formation assay investigating cancer cell growth in the lung cancer cell line Calu3 , the liver cancer cell line HepG2 , and the pancreatic cancer cell line Panel, in which TSPYL5 gene was inhibited, and the results of Western blotting performed to investigate the expressions of the cancer stem cell markers ALDH1A1 and ALDH1A3.

[Best Mode]

Hereinafter, the present invention is described in detail .

The present invention provides a pharmaceutical composition for preventing and inhibiting cancer metastasis comprising the TSPYL5 (testis-specific protein, Y-encoded- like 5) expression or activity inhibitor as an active ingredient .

The said TSPYL5 preferably has the amino acid sequence represented by SEQ. ID. NO: 1, but not always limited thereto.

The TSPYL5 expression inhibitor is preferably the antisense nucleotide, small interfering RNA, or shRNA (short hairpin RNA) that binds complementarily to TSPYL5 mRNA, and the TSPYL5 activity inhibitor is preferably selected from the group consisting of the compounds, peptides, peptide mimetics, and antibodies which are complementarily binding to TSPYL5 protein, but not always limited thereto.

The said siRNA is composed of the sense sequence in 15-30 mer selected from the nucleotide sequences of mRNA of the gene (SEQ. ID. NO: 2) encoding human TSPYL5 protein and the antisense sequence complementarily binding thereto. At this time, the sense sequence herein is preferably composed of 25 nucleotides and more preferably composed of the nucleotide sequence represented by SEQ. ID. NO: 17 or NO: 18, but not always limited thereto.

The said shRNA indicates the double-stranded RNA having the hair pin structure containing a loop region comprising 2-10 nucleotides, and the nucleotides in the loop can be selected among the nucleotides well known to those in the art (Proc. Natl. Acad. Sci. US A 99(8): 5515- 5520, 2002; Nature Biotechnology 20: 505-508, 2002; Nature Biotechnology 20 : 500-505, 2002; Nat Cell Biol. 5:489-490, 2003; Proc. Natl. Acad. Sci . USA 99 ( 9) : 6047 -6052 , 2002). The double stranded region of shRNA can be the same as that of siRNA.

The said antisense nucleotide, as defined by Watson- Click base-pair, is bound to the complementary sequence of DNA, premature mRNA, or mature mRNA, by which it interrupts the flow of genetic information from DNA to protein. The target specific antisense nucleotide is characterized by exceptional multi-functions. Since the antisense nucleotide is a long chain in monomer, it can be easily synthesized to the target RNA sequence. Many of recent studies proved the usability of the antisense nucleotide as a biochemical tool usable for the study of a target protein (Rothenberg et al . , J. Natl. Cancer Inst., 81:1539-1544, 1999) . According to the recent advancement in the study of oligonucleotide chemistry and synthesis of nucleotide having improved cell adhesion activity, target binding affinity, and neclease resistance, the antisense nucleotide can be a novel type of inhibitor.

The said peptide mimetics is a peptide or non-peptide that inhibits the binding domain of TSPYL5 , that is it can inhibit the activity of TSPYL5. The major residue of a non-hydrolysable peptide analog can be generated by using β-turn dipeptide core (Nagai et al . Tetrahedron Lett., 26:647, 1985), keto-methylene pseudopeptides (Ewenson et al . J Med Chem 29:295, 1986; and Ewenson et al . in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, IL, 1985) , azepine (Huffman et al . in Peptides: Chemistry and Biology, G.R. Marshall ed. , ESCOM Publisher: Leiden, Netherlands, 1988), benzodiazepine (Freidinger et al . in Peptides; Chemistry and Biology, G.R. Marshall ed. , ESCOM Publisher: Leiden, Netherlands, 1988), β-amino alcohol (Gordon et al . Biochem Biophys Res Commun 126:419 1985), and substituted gamma-lactam ring (Garvey et al . in Peptides: Chemistry and Biology, G.R. Marshell ed. , ESCOM Publisher: Leiden, Netherlands, 1988) .

The TSPYL5 expression or activity inhibitor preferably inhibits the cancer stem cell growth and metastasis, but not always limited thereto.

The cancer herein is preferably selected from the group consisting of liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head/neck cancer, uterine cancer, colorectal cancer, lung cancer, ovarian cancer, rectal cancer, esophageal cancer, small bowel cancer, anal cancer, fallopian tube carcinoma, endometrial carcinoma, uterine cervical carcinoma, vaginal carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, and central nervous system tumor, but not always limited thereto.

In a preferred embodiment of the present invention, the present inventors investigated the expression of TSPYL5 after the fractionated irradiation. As a result, it was confirmed that the expressions of the cancer stem cell marker and the cancer stem cell marker related proteins, and the expression of TSPYL5 were increased by the irradiation. The expression of TSPYL5 protein was higher with the three times divided fractionated irradiation with 2 Gy than the single irradiation (2 Gy, 4 Gy) (see Figure 1) .

Cancer cell invasion and migration over the fractionated irradiation were investigated. As a result, the cell invasion and migration in A549 cell line was increased after the fractionated irradiation, but the expression of E-cadherin, the epithelial cell marker, one of EMT markers, was reduced after the fractionated irradiation. The expressions of N-cadherin and Snail, the mesenchymal cell markers, were increased by the fractionated irradiation (see Figure 2) .

ALDH1 active cells and ALDH1 inactive cells, separated from A549 cell line, were compared. As a result, ALDH1 active cells grew well, but ALDH1 inactive cells did not grow well. In the meantime, the expressions of the cancer stem cell markers ALDH1A1, ALDH1A3 , Cd44, and CD133, and the expressions of TSPYL5 gene and protein were all increased in ALDH1 active cells, compared with those in ALDH1 inactive cells, On the other hand, the expression of PTEN was higher in ALDH1 inactive cells than in ALDH1 active cells (see Figure 3) .

It was also investigated how TSPYL5 expression was related to the cancer stem cell marker ALDH1. As a result, when TSPYL5 was over-expressed, the cancer stem cell marker ALDH1 expression was increased and cell proliferation was also accelerated. In the meantime, when TSPYL5 expression was inhibited, the cancer stem cell marker ALDH1 expression was decreased and cancer cell growth was also restrained (see Figure 4) . Metastasis through cell invasion/migration over TSPYL5 expression was also investigated. As a result, when TSPYL5 expression was inhibited, metastasis was also inhibited, while when TSPYL5 was over-expressed, metastasis was increased (see Figure 5) .

When TSPYL5 expression was inhibited, the size of sphere, the major characteristics of tumorigenic transformation, was comparatively small, indicating the cancer cell growth was accordingly inhibited. when TSPYL5 was over-expressed, the size of sphere, the major characteristics of tumorigenic transformation, was enlarged, indicating that the cancer cell growth was accelerated thereby (see Figure 6) .

The correlation of ALDHl expression and TSPYL5 was also investigated. As a result, TSPYL5 expression was not changed by the inhibition of ALDHl expression, but the cell growth was significantly inhibited. TSPYL5 expression was not changed by the over-expression of ALDHl, either, but the cell growth was increased. From the above results, it was confirmed that TSPYL5 regulates ALDHl at a higher level (see Figure 7) . When ALDHl expression was inhibited, the cell invasion/migration was suppressed to inhibit metastasis, the expressions of the mesenchymal cell protein markers N-cadherin, Vimentin, Twist, Snaill and Slug, members of EMT marker family, were reduced, and the expression of E-cadherin, the epithelial cell marker, was increased. When ALDHl was over-expressed, the cell invasion/migration was increased to increase metastasis, the expressions of the mesenchymal cell protein markers N- cadherin, Vimentin, Twist, Snaill and Slug, members of EMT marker family, were increased, but the expression of E- cadherin, the epithelial cell marker, was decreased (see Figure 8) .

The expression of TSPYL5 was inhibited in Calu3 (adenocarcinoma cell line) , HepG2 (liver cancer cell line) , and Panel (pancreatic cancer cell line) , in which TSPYL5 is usually rich, in addition to the non-small cell lung cancer cell line A549 examined hereinbefore. Then, the size of sphere, the major characteristics of tumorigenic transformation, was investigated. As a result, like in A549, the size of sphere was comparatively small in the adenocarcinoma cell line Calu3 , the liver cancer cell line HepG2, and the pancreatic cancer cell line Panel, when TSPYL5 expression was inhibited. And, it was confirmed that the cancer stem cell growth was inhibited by the decrease of the expression of ALDH1, the major cancer stem cell marker (see Figure 9) .

The present inventors performed the fractionated irradiation on the non-small cell lung cancer cell line A549. As a result, the expressions of TSPYL5, and the cancer stem cell markers ALDH1 and CD44 were significantly increased. In the meantime, the inhibition of TSPYL5 expression or activity led to the inhibition of the expressions of the cancer stem cell markers ALDH1 and CD44, and accordingly the function of EMT, a major pathway of the cancer stem cell growth and metastasis, was significantly reduced. So, the inhibition of TSPYL5 expression or activity resulted in the inhibition of tumorigenic cell growth, so that the TSPYL5 expression or activity inhibitor can be effectively used as a pharmaceutical composition for inhibiting cancer metastasis in various kinds of cancers. The composition of the present invention can additionally include carriers, excipients and diluents generally used for the preparation of a pharmaceutical composition .

The composition of the present invention can be administered orally or parenterally . The parenteral administration herein can be performed by external application, intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection, but not always limited thereto.

The composition of the present invention can be formulated by the conventional method as powders, granules, tablets, capsules, suspensions, emulsions, syrups, liquids, water- insoluble excipients, suspensions, aerosols, external preparations, suppositories, and sterilized injections. The carriers, excipients and diluents are exemplified by lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate , propylhydroxybenzoate , talc, magnesium stearate and mineral oil. Formulations can be prepared by using generally used excipients or diluents such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactant. Solid formulations for oral administration are tablets, pills, powders, granules and capsules. These solid formulations are prepared by mixing one or more suitable excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. Except for the simple excipients, lubricants, for example magnesium stearate, talc, etc, can be used. Liquid formulations for oral administrations are suspensions, solutions, emulsions and syrups, and the above-mentioned formulations can contain various excipients such as wetting agents, sweeteners, aromatics and preservatives in addition to generally used simple diluents such as water and liquid paraffin. Formulations for parenteral administration are sterilized aqueous solutions, water- insoluble excipients, suspensions, emulsions, lyophilized preparations, suppositories and injections. Water insoluble excipients and suspensions can contain, in addition to the active compound or compounds, propylene glycol, polyethylene glycol, vegetable oil like olive oil, injectable ester like ethylolate, etc. Suppositories can contain, in addition to the active compound or compounds, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, etc.

The effective dosage of the composition of the present invention can be determined according to weight and condition of a patient, severity of a disease, preparation of a drug, administration pathway and time. The effective dosage of the composition of the present invention is preferably 0.0001 ~ 1 g/kg per day, and more preferably 0.001 ~ 200 mg/kg per day, but not always limited thereto. The administration frequency can be once a day or a few times a day. The above dosage cannot limit the scope of the present invention in any way.

The present invention also provides a method for preventing cancer metastasis containing the step of administering a pharmaceutically effective dose of the TSPYL5 expression or activity inhibitor to a subject having cancer .

The present invention further provides a method for inhibiting cancer metastasis containing the step of administering a pharmaceutically effective dose of the TSPYL5 expression or activity inhibitor to a subject having cancer .

The TSPYL5 expression inhibitor is preferably the antisense nucleotide, small interfering RNA, or shRNA (short hairpin RNA) that binds complementarily to TSPYL5 mRNA, and the TSPYL5 activity inhibitor is preferably selected from the group consisting of the compounds, peptides, peptide mimetics, and antibodies binding TSPYL5 complementarily, but not always limited thereto.

The cancer herein is preferably selected from the group consisting of liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head/neck cancer, uterine cancer, colorectal cancer, lung cancer, ovarian cancer, rectal cancer, esophageal cancer, small bowel cancer, anal cancer, fallopian tube carcinoma, endometrial carcinoma, uterine cervical carcinoma, vaginal carcinoma, Hodgkin ' s disease, prostate cancer, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, and central nervous system tumor, and more preferably selected from the group consisting of breast cancer, uterine cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, stomach cancer, and pancreatic cancer, but not always limited thereto .

The present inventors performed the fractionated irradiation on the non-small cell lung cancer cell line A549. As a result, the expressions of TSPYL5, and the cancer stem cell markers ALDH1 and CD44 were significantly increased. In the meantime, the inhibition of TSPYL5 expression or activity led to the inhibition of the expressions of the cancer stem cell markers ALDH1 and CD44, and accordingly the function of EMT, a major pathway of the cancer stem cell growth and metastasis, was significantly reduced. So, the inhibition of TSPYL5 expression or activity resulted in the inhibition of tumorigenic cell growth, so that the TSPYL5 expression or activity inhibitor can be effectively used in the treatment of a variety of cancers, precisely can be hired by the method for preventing or inhibiting cancer metastasis containing the step of administering the inhibitor to a subject having cancer .

The cancer herein is preferably selected from the group consisting of liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head/neck cancer, uterine cancer, colorectal cancer, lung cancer, ovarian cancer, rectal cancer, esophageal cancer, small bowel cancer, anal cancer, fallopian tube carcinoma, endometrial carcinoma, uterine cervical carcinoma, vaginal carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, and central nervous system tumor, and more preferably selected from the group consisting of breast cancer, uterine cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, stomach cancer, and pancreatic cancer, but not always limited thereto.

The kit herein can additionally contain a substrate color-reacting with an enzyme, and a washing buffer or eluent to eliminate non-conjugated proteins with leaving conjugated markers only. The sample for the analysis includes such biological materials, which are suitable for the identification of a disease-specific polypeptide which is different from a normal polypeptide, as serum, urine, tear, and saliva, etc. Preferably, the sample indicates a biological liquid sample such as blood, serum, and plasma, and more preferably serum. The sample can be prepared to increase detection sensitivity, for example a serum sample obtained from a patient can be pre-treated by anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, sequential extraction, or gel electrophoresis, but not always limited thereto .

In this invention, it was confirmed that the expression of the cancer stem cell marker ALDH1 was increased by the over-expression of TSPYL5 in the lung cancer cell line. On the other hand, the expression of the cancer stem cell marker ALDH1 was decreased by the inhibition of TSPYL5 using siRNA. Therefore, a sample that demonstrates the down-regulation of ALDH1, either in expression or in activity, according to the inhibition of TSPYL5 expression or activity can be used for a kit for inhibiting the cancer stem cell growth in various types of cancers .

The cancer herein is preferably selected from the group consisting of liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head/neck cancer, uterine cancer, colorectal cancer, lung cancer, ovarian cancer, rectal cancer, esophageal cancer, small bowel cancer, anal cancer, fallopian tube carcinoma, endometrial carcinoma, uterine cervical carcinoma, vaginal carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, and central nervous system tumor, and more preferably selected from the group consisting of breast cancer, uterine cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, stomach cancer, and pancreatic cancer, but not always limited thereto .

In this invention, it was confirmed that the expression of the cancer stem cell marker ALDH1 was increased by the over-expression of TSPYL5 in the lung cancer cell line. On the other hand, the expression of the cancer stem cell marker ALDH1 was decreased by the inhibition of TSPYL5 using siRNA. Therefore, a sample that demonstrates the down-regulation of ALDH1, either in expression or in activity, according to the inhibition of TSPYL5 expression or activity can be used for a method for inhibiting the cancer stem cell growth in various types of cancers .

The present invention also provides a method for screening a cancer metastasis inhibitor candidate comprising the following steps :

1) preparing the cell line expressing TSPYL5;

2) treating the sample material to the cell line of step 1) ;

3 ) measuring the expression or activity of ALDH

(aldehyde dehydrogenase) in the cell line; and

4) selecting the sample material that could inhibit the expression or activity of ALDH, compared with the expression or activity thereof in the control not-treated with the sample material. In addition, the present invention provides a method for screening a cancer stem cell growth inhibitor candidate comprising the following steps:

1) preparing the cell line expressing TSPYL5 ;

2) treating the sample material to the cell line of step 1) ;

3) measuring the expression or activity of ALDH in the cell line; and

4) selecting the sample material that could reduce the expression or activity of ALDH, compared with the expression or activity thereof in the control not-treated with the sample material.

In the above method, the cell line expressing TSPYL5 of step 1) is preferably constructed by irradiating a cancer cell line.

The irradiation herein is preferably performed by the single irradiation with 2 Gy or 4 Gy or by the fractionated irradiation with 2 Gy, 2 - 5 times, and more preferably performed by the three divided fractionated irradiation with 2 Gy.

In this invention, it was confirmed that the expression of the cancer stem cell marker ALDH1 was increased by the over-expression of TSPYL5 in the lung cancer cell line. On the other hand, the expression of the cancer stem cell marker ALDH1 was decreased by the inhibition of TSPYL5 using siRNA. Therefore, a sample that demonstrates the down-regulation of ALDH1, either in expression or in activity, according to the inhibition of TSPYL5 expression or activity can be used for screening a cancer metastasis inhibitor candidate and a cancer stem cell growth inhibitor candidate in various types of cancers iMode for Invention]

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention. Example 1: Investigation of TSPYL5 gene expression over the fractionated irradiation in the lung cancer cell line

<!-!> Culture of the non-small cell lung cancer cell line and measurement of TSPYL5 expression over the fractionated irradiation

To confirm the increase of TSPYL5 expression by the fractionated irradiation, the lung cancer cell line was irradiated (radiation source; ⁶⁰C₀) at the Gamma-ray Facility of Korea Atomic Energy Research Institute (KAERI) , followed by Western blotting.

Particularly, the lung cancer cell line A549 (American

Type Culture Collection, USA) was cultured in RPMI-1640 (Hyclone) supplemented with 10% fetal bovine serum (Hyclone) and 100 Unit/ml penicillin-streptomycin solution (Hyclone) . The cells were distributed in 75 flasks at the density of lxlO⁶ cells/flask and then cultured in a 37^°C , 5% C0₂ incubator. Upon completion of the culture, the cells were irradiated at the dose of 2 Gy three times at two days intervals. On the final irradiation (third irradiation) day, the control A549 cells were irradiated once at the dose of 2 Gy and 4 Gy. The irradiated cells were cultured in a 37^°C , 5% C0₂ incubator for 7 days. The cells of each group were collected, to which 50 ul of lysis buffer was added, followed by reaction at 4 ^°C for 30 minutes. Pellet and the supernatant were separated- by centrifugation at 13,000 rpm at 4^°C . The protein was quantified from the supernatant by using a protein quantification kit (Sigma) and 40 ug of each protein was loaded on SDS-gel. The protein loaded on SDS-gel was transferred onto nitrocellulose membrane, followed by reaction in BSA buffer at room temperature for 30 minutes, by which non-specific antibody conjugation was prevented. The membrane was reacted with the primary antibodies ALDH1A1, ALDH1A3 (Abeam), TSPYL5, PTEN, β-catenin, Jaggedl (Santa Cruz), Oct4 (Millipore) , CD44, Sox2, and -actin(Cell Signaling) diluted with PBS (1:1000) for 4 hours. Then, the membrane was reacted with the secondary antibodies anti -Rabbit and anti-Mouse (Cell Signaling) diluted with PBS (1:10000) for 1 hour. The nitrocellulose membrane was washed with PBS 5 times, followed by photosensitization on the film using a detection solution. As a result, as shown in Figure 1A, it was confirmed that the expressions of the cancer stem cell marker and the cancer stem cell marker related proteins, and the expression of TSPYL5 were increased by the irradiation. The expression of TSPYL5 protein was higher with the three times divided fractionated irradiation with 2 Gy than the single irradiation (2 Gy, 4 Gy) . As a result, as shown in Figure 1A, the expressions of TSPYL5, the cancer stem cell marker, and the related protein were all increased by the irradiation. The cancer stem cell marker and the related proteins were also over-expressed by the fractionated irradiation (Figure 1A) .

<l-2> Investigation of TSPYL5 expression and location after the fractionated irradiation by using intracellular staining

To confirm the increase of TSPYL5 expression by the fractionated irradiation, the lung cancer cell line was irradiated with gamma-ray (radiation source; ⁶⁰C₀) at the Gamma-ray Facility of Korea Atomic Energy Research Institute (KAERI) , followed by intracellular staining using immunofluorescence .

The lung cancer cell line A549 and the A549 treated with the fractionated irradiation were cultured on the dish with cover glass at the density of 1x10^s cells/dish for 24 hours. The cells were fixed with 4% paraformaldehyde solution for 20 minutes and the cover glass was washed with PBS three times, followed by reaction in 0.3 ~ 0.5% Triton- X 100 solution for 5 minutes. The glass was washed again with PBS three times. The cells were reacted with 1% BSA for 20 minutes, by which non-specific antibody conjugation was prevented. The cells were reacted with the primary antibody TSPYL5 (Santa Cruz) and the cancer stem cell marker ALDH1A1 (Cell Signaling) diluted with PBS (1:1000) for 2 hours . Then, the cells were reacted with the secondary antibody anti-Rabbit (Cell Signaling) diluted with PBS (1:1000) for 1 hour. The cells were washed with PBS three times and the nucleus was stained with DAPI solution for 5 minutes. The TSPYL5 expression and location were observed under fluorescence microscope.

As a result, as shown in Figure IB, the expressions of TSPYL5 and ALDH1A1 were increased in the A549 cells treated with the fractionated irradiation and TSPYL5 which used to be found in cytoplasm was up-regulated in the nucleus by the fractionated irradiation (Figure IB) .

Example 2 : Investigation of cancer cell metastasis over the fractionated irradiation using migration/invasion assay

Cancer cell migration and invasion over the fractionated irradiation were investigated by using raatrigel kit and by Western blotting.

Particularly, to measure the migration/invasion of cancer cells over the fractionated irradiation, 0.8 um pore size transwell (Falcon, USA) was used. For the migration assay, the lung cancer cell line A549 and the A549 treated with the fractionated irradiation were cultured in 100 ul of serum-free RPMI 1640 at the density of 5xl0⁴ cells in the upper chamber of transwell. The lower chamber was filled with 500 ul of RPMI 1640 supplemented with 7% FBS . Then, the two chambers were combined. The cells were maintained in a 37°C, 5% C0₂ incubator for 40 hours and then the membrane in the upper chamber was wiped out with cotton swabs, followed by staining with crystal violet, which was then observed under microscope.

Invasion assay was also performed by the same manner as described above for the migration assay except that 100 ul of Matrigel (20 ug/well; BD GBiosciences) was pre-loaded in the upper chamber of transwell for coating. The cells stained with crystal violet were eluted by using 500 ul of 10% acetic acid, and OD_SOo measured to determine the relative value of invasion/migration of A549 cells.

As a result, as shown in Figure 2A, the migration and invasion was more significant in A549 cells treated with the fractionated irradiation (Figure 2A) .

The cells treated with the fractionated irradiation were cultured for 7 days, followed by Western blotting by the same manner as described in Example <1-1> by using the primary antibodies N-cadherin (Santa Cruz) , E-cadherin, β- actin (Cell Signaling), and Snail (Abeam) , which have been used as the epithelial to mesenchymal transition (EMT) markers, and the secondary antibody anti-Rabbit (Cell Signaling) .

As a result, as shown in Figure 2B, the expression of E-cadherin, the epithelial cell marker, one of EMT markers, was reduced after the fractionated irradiation. The expressions of N-cadherin and Snail, the mesenchymal cell markers, were increased by the fractionated irradiation (Figure 2B) .

Therefore, it was confirmed that the fractionated irradiation was closely related to EMT, and accordingly the cell migration and invasion were increased by the fractionated irradiation.

Example 3 : Separation of ALDHl active cells and ALDHl inactive cells from the ^' lung cancer cell line using Aldefluor

<3-l> Separation of ALDHl active cells and ALDHl inactive cells from the lung cancer cell line

To investigate the correlation of TSPYL5 and the lung cancer stem cell growth in A549 through ALDHl activity, the cells were stained with Aldefluor and then the cancer stem cells were separated therefrom using FACS (fluorescence activated cell sorter) .

Particularly, to separate ALDH1 active and inactive cells from A549 cells, Aldefluor (Stem Cell) was used. This product can up-regulates ALDH higher in the stem cells than in the regular cells based on the activity of aldehyde dehydrogenase (ALDH) . So, it is useful to separate live stem cells because, according to the enzyme activity, the cells having damaged cell membrane are excluded in counting by that . It is also advantageous because it has no toxicity and is convenient in use. 25 ul of DMSO (dimethylsulfoxide) was added to the dried Aldefluor reagent, followed by reaction at room temperature for 1 minute, resulting in the activation of the enzyme which could be confirmed when the color of the reagent turned from red-orange to light-yellow. At that moment, 25 ul of 2 M hydrochloric acid was added thereto, followed by reaction at room temperature for 15 minutes. ^' 360 ul of Aldefluor assay buffer was added thereto, which was stored in a refrigerator, resulting in the preparation of Aldefluor substrate. The prepared A549 cells were lysed, followed by centrifugation to separate the supernatant. 1 ml of Aldefluor assay buffer was added to the supernatant, making the density of lxlO⁶ cell/ml. Two empty tubes were prepared. 5 ul of the activated Aldefluor substrate was added to the lysate, followed by mixing. 500 ul of the mixture was loaded in the control tube . 5 ul of DEAB (diethylaminobenzaldehyde) solution was added only to the control tube. After inducing reaction at 37^°C for 30 minutes each, the supernatant was eliminated by centrifugation . 500 ul of Aldefluor assay buffer was added thereto, followed by reaction at 4^°C for 24 hours. When the negative charged ALDH-substrate BAAA was invaded into the live cells by passive diffusion, it was converted into BAA- by the intracellular ALDH with emitting fluorescence. Those cells emitting fluorescence were separated as active cells and those cells not-emitting fluorescence were separated as inactive cells, by FACS (Figure 3A) .

<3-2> Colony formation assay with the separated ALDHl active cells and ALDHl inactive cells

To compare the cell growth among A549 and ALDHl active cells and ALDHl inactive cells, both separated from A549, colony formation assay was performed with them.

Particularly, ALDHl active cells and ALDHl inactive cells were inoculated in 35 mm plates at the density of lxlO³ cells/plate, followed by culture in a 37^°C C0₂ incubator for 8 days. The cells were stained with crystal violet (0.5%) and then washed several times with PBS, followed by observation under microscope.

As a result, as shown in Figure 3B, ALDH1 active cells grew well, but ALDH1 inactive cells were not so much grown (Figure 3B) .

<3-3> The expressions of TSPYL5 and the cancer stem cell marker in ALDH1 active cells and ALDH1 inactive cells

To investigate the expressions of TSPYL5 and the cancer stem cell markers CD44 and CD133 in ALDH1 active and inactive cells separated from A549, PCR and Western blotting were performed.

Particularly, the separated ALDH1 active and inactive cells were mixed in 1 ml of trizol, to which 200 ul of chloroform was added. After well mixing for 5 minutes, the mixture was centrifuged at 4^°C for 10 minutes. 200 ul of the supernatant was transferred into a new tube, to which 500 ul of isopropanol was added, followed by reaction at room temperature for 10 minutes. Centrifugation was performed at 4^°C and the supernatant was discarded. The precipitate was washed with DEPC (diethylpyrocarbonate) solution containing 75% ethanol, followed by centrifugation again to eliminate the supernatant. The resultant precipitate was dissolved in DEPC solution, followed by quantification. cDNA was synthesized from 1 ug of RNA by using oligo dT cDNA kit (Intron) (45 ^°C, lhour; 95 ^°C , 5 minutes) . Amplification was performed using the primers presented in table 1 and I-taq polymerase (Intron) as follows: predenaturation at 94 ^°C for 5 minutes, denaturation at 94 ^°C for 30 seconds, annealing at 56 °C for 30 seconds, polymerization at 72^°C for 30 seconds, 30 cycles from denaturation to polymerization, and final extension at 72 ^°C for 10 minutes. The PCR product was electrophoresed on 1% agarose gel.

As a result, as shown in Figure 3C, the expressions of the cancer stem cell markers ALDH1A1 , ALDH1A3 , CD44, and CD133, and the expression of TSPYL5 were increased more in ALDH1 active cells than in ALDH1 inactive cells. On the other hand, the expression of PTEN was higher in ALDH1 inactive cells than in ALDH1 active cells (Figure 3C) .

50 ul of lysis buffer was added to the cells, followed by reaction at 4 ^°C for 30 minutes. Centrifugation was performed at 4^°C. 40 ul of each protein obtained from the supernatant by using protein quantification kit (Sigma) was loaded on SDS-gel. Protein expression was confirmed using the primary antibodies of ALDH1A1, ALDH1A3 (Abeam) , TSPYL, PTEN (Santa Cruz), CD44, β-actin (Cell Signaling) and CD133 (Biorbyt) by the same manner as described in Example <1-1>.

As a result, as shown in Figure 3D, the expressions of the cancer stem cell markers ALDH1A1, ALDH1A3 , CD44, and CD133, and the expression of TSPYL5 were increased in ALDH1 active cells, which was consistent with the result of PCR, but the expression of PTEN was decreased therein (Figure 3D) .

Therefore, it was confirmed that TSPYL5 were rich in the cancer stem cell marker ALDH1 active cells and is closely related to the growth of lung cancer cell.

[Table l]

SEQ.

ATGGACAAGTTTTGGTG

CD44_F Forward ID.

GCACGCA

NO: 9

CD44 57^°C/30

SEQ.

TCACCCCAATCTTCATG ID.

CD44_R Reverse

TCCACAT NO:

10

SEQ.

TTCGGCTCTCCAGGAAG ID.

TSPYL5_F Forward

TTT NO:

TSPYL5 11 57^°C/30

SEQ.

GGGGATGGTTCTGAAAT ID.

TSPYL5_R Reverse

GCT NO:

12

SEQ.

TGTGGTCTGCCAGCTAA ID.

PTEN_F Forward

AGG NO:

PTEN 13 57^°C/30

SEQ.

CACACAGGTAACGGCTG ID.

PTEN_R Reverse

AGG NO:

14

SEQ.

AAGGGTCATCATCTCTG ID.

GAPDH_F Forward

CCC NO:

GAPDH 15 56^°C/25

SEQ.

AGGGGTGCTAAGCAGTT ID.

GAPDH_R Reverse

GGT NO:

16 Example 4: Correlation of TSPYL5 and the cancer stem cell marker ALDH1

To confirm the correlation of TSPYL5 expression and the cancer stem cell marker ALDH1, the present inventors induced the over-expression of TSPYL5 and the inhibition of TSPYL5 to regulate the expression of TSPYL5.

Particularly, to inhibit the expression of TSPYL5 using TSPYL5 siRNA in A549 which usually expresses TSPYL5 abundantly, TSPYL5 stealth-RNAi in 25 mer [forward primer: 5 ' -AAAGGUAGAACUGCAAGGGAUUGGG-3¹ (SEQ. ID. NO: 17), Reverse primer : 5 ' -CCCAAUCCCUUGCAGUUCUACCUUU-3 ' (SEQ . ID . NO : 18 ) , Invitrogen] was used. To insert the siRNA into the cell, 100 nM of RNAi was treated to 2xl0⁵ A549 cells, and the cells were reacted in penicillin-streptomycin solution (Hyclone) free medium for 4 - 6 hours in the presence of lipofectamine RNAi MAX (Invitrogen) . For the negative control (siControl) , Scrambled StealthTM RNA molecule was used. Then, the medium was replaced with the medium containing penicillin- streptomycin, followed by culture for 72 hours. In the meantime, to over-express TSPYL5 gene in H460, TSPYL5 over-expressing clone was constructed and inserted in H460. Amplification was performed using the forward primer [5 ' -CTTAAGCTTATGAGCGGCCGAAGTCGG-3^■ (SEQ . ID . NO: 19)] and the reverse primer [5'- TGGAATTCGTGTTGGATTGGCTCACCCC-3 ' (SEQ. ID. NO: 20)] designed to contain Hindlll restriction enzyme recognition site at 5' -end and EcoRI restriction enzyme recognition site at 3'- end as follows: predenaturation at 94 ^°C for 5 minutes, denaturation at 94 ^°C for 1 minute, annealing at 56 ^°C for 1 minute, polymerization at 72^°C for 1.5 minute, 30 cycles from denaturation to polymerization, and final extension at 72 ^°C for 5 minutes. As a result, 1253 bp TSPYL5 gene was obtained. The PCR product obtained above and pcDNA3.1 vector (Invitrogen, USA) were treated with restriction enzymes and ligated by ligase, resulting in the construction of fibulin-3 over-expressing pcDNA3.1/TSPYL5 vector. 2 ug of the said TSPYL5 over-expressing vector was introduced in 2xl0⁵/ml of H460 cells by using lipofectamine 2000 in penicillin-streptomycin free medium, followed by reaction for 4 - 6 hours. Then, the medium was replaced with the fresh medium supplemented with 100 units/ml penicillin-streptomycin, followed by culture for 48 hours.

<4-l> Expression of the cancer stem cell marker ALDHl according to the over-expression and inhibition of TSPYL5

To confirm the over-expression of TSPYL5 in H460 cells and the inhibition of TSPYL5 in A549 cells, the present inventors performed PCR using TSPYL5 and ALDHl primers and Western blotting using TSPYL5 and ALDHl antibodies by the same manner as described in Example <3-3>. As a result, as shown in Figure 4A, when TSPYL5 was over-expressed in H460 cells, the cancer stem cell marker ALDH1 was up-regulated. When TSPYL5 expression was inhibited in A549 cells, the cancer stem cell marker ALDH1 was down-regulated (Figure 4A) .

Therefore, it was confirmed that the regulation of TSPYL5 expression was closely related to the regulation of the cancer stem cell markers ALDH1A1 and ALDH1A3 genes and proteins .

<4-2> Investigation of cancer cell growth over the over- expression and inhibition of TSPYL5

The present inventors performed colony formation assay with A549, A549 with TSPYL5 inhibited, the control H460, and H460 with TSPYL5 over-expressed by the same manner as described in Example <3-2>.

As a result, as shown in Figure 4B, the inhibition of TSPYL5 in A549 cells resulted in the suppression of cell growth. In the meantime, the over-expression of TSPYL5 in H460 cells increased the cell proliferation (Figure 4B) .

<4-3> Expression of ALDH according to the over-expression and inhibition of TSPYL5

The present inventors stained A549, A549 with TSPYL5 inhibited, the control H460, and H460 with TSPYL5 over- expressed with Aldefluor by the same manner as described in Example <3-l>, followed by FACS .

As a result, as shown in Figure 4C, the cell growth was inhibited in A549 cells with TSPYL5 inhibited, while the cell growth was increased in H460 cells with TSPYL5 over-expressed (Figure 4C) .

<4-4> Separation of ALDHl active cells and ALDH expression pattern over the inhibition of TSPYL5

The present inventors inhibited TSPYL5 in ALDH (+ , high) active cells separated in Example <3-l> by the same manner as described in Examples <4-l> and <4-2>, followed by PCR using TSPYL5 and ALDHl primers and Western blotting using TSPYL5 and ALDHl antibodies, and colony formation assay as well.

As a result, as shown in Figure 4D, TSPYL5 and ALDH genes and proteins were suppressed in ALDH active cells, the cancer stem cells, when TSPYL5 was inhibited, and accordingly the cancer cell growth was also inhibited (Figure 4D) .

Example 5: Investigation of metastasis using the over- expression and inhibition of TSPYL5

<5-l> Confirmation of EMT marker using the over-expression and inhibition of TSPYL5 The present inventors performed Western blotting and the experiment using matrigel kit with the lung cancer cell line A549, A549 with TSPYL5 inhibited, the control H460, and H460 with TSPYL5 over-expressed, in order to investigate how the cell migration/invasion was affected by the regulation of TSPYL5 gene.

Particularly, A549, A549 with TSPYL5 inhibited, the control H460, and H460 with TSPYL5 over-expressed were cultured by the same manner as described in Example <4-l>. Western blotting was performed by the same manner as described in Example <1-1> using the primary antibodies of N-cadherin, Twist (Santa Cruz) , E-cadherin, β-actin (Cell Signaling) , and Snail (Abeam) , which have been used as the EMT markers, and the secondary antibody anti-Rabbit and anti-Mouse (Cell Signaling) .

As a result, as shown in Figures 5A and 5C, the expression of E-cadherin, the epithelial cell marker, one of EMT markers, was increased in A549 with TSPYL5 inhibited, while the expressions of N-cadherin, Vimentin, Twist, and Snail, the mesenchymal cell markers, were inhibited (Figure 5A) . In the meantime, the expressions of those mesenchymal cell markers among the EMT marker family were increased in H460 with TSPYL5 over-expressed (Figure 5C) . <5-2> Investigation of cancer cell metastasis according to the over-expression and inhibition of TSPYL5

The present inventors performed the migration/invasion assay using transwell (Falcon, USA) by the same manner as described in Example 2 , in order to measure the cancer cell metastasis.

Particularly, A549, A549 with TSPYL5 inhibited, the control H4.fi0, and H460 with TSPYL5 over-expressed were cultured, which were distributed in the upper chamber of transwell (5xl0⁴ cells) . The lower chamber was filled with 500 ul of RPMI 1640 supplemented with 7% FBS . Another transwell chamber was pre-treated with 100 ul of matrigel (20 ug/well: BD GBiiosciences ) for coating. The cells were maintained in a 37^°C , 5% C0₂ incubator for 40 hours and then the membrane in the upper chamber was stained with crystal violet, followed by observation under microscope to count the number of stained cells.

As a result, as shown in Figures 5B and 5D, the migration/invasion capacity was reduced by the inhibition of TSPYL5 gene, while the migration/invasion of cell was increased by the over-expression of TSPYL5 gene (Figures 5B and 5D) .

Therefore, it was confirmed that the expressions of EMT markers and the migration/invasion capacity could be changed by the regulation of TSPYL5 gene. Example 6 : Confirmation of the changes in sphere formation by the regulation of TSPYL5 expression

The tumorigenic cells are growing anchorage- independently on agar plate. The anchorage-independent cell growth is a key phenomenon to determine the possibility of tumorigenic transformation of cells. TSPYL5 expression was inhibited by using shRNA in conditional media and then the anchorage independent cell growth was compared .

<6-l> shRNA lentivirus transduction for TSPYL5 inhibition

To investigate the correlation of TSPYL5 and sphere formation, the major characteristics of tumorigenic transformation of cancer cells, the present inventors first prepared shRNA TSPYL5 for the continuing inhibition of TSPYL5 expression.

Particularly, lxlO⁶ 293T cells (Korea Institute of Radiological & Medical Sciences, Seoul) were distributed in 60 mm plate, and the cells were transfected with 1 ug/ul of the virus early gene vector (pLPl, pLP2, pLP/VSVG; Invitrogen) , 2 ug/ul of shRNA (Table 2, SHCLNG-NM_033512 ; Sigma-Aldrich MISSION shRNA library) , and lipofectamine 2000 in penicillin-streptomycin free medium. 12 hours later, the medium were replaced with the medium containing 100 units/ml penicillin-streptomycin, followed by culture for 24 hours. The culture fluid containing the virus particles was treated to A549 cells separated as single cells .

[Table l\

<6-2> Investigation of sphere formation according to the inhibition of TSPYL5 expression in the lung cancer cell line

The present inventors investigated sphere formation according to the inhibition of TSPYL5 expression. Particularly, 2xl0⁴ A549 cells were suspended in DMEM (Invitrogen) containing stem cell-permissive medium. DMEM- 12 (Invitrogen) was mixed with 20 ng/ml EGF, 20 ng/ml basic fibroblast growth factor (bFGf ) , and B27 serum- free supplement (50x; Invitrogen) , which was loaded in 60 mm plate pre-coated with 0.8% agarose. Then, the suspended cells above were loaded in the plate for culture. As for the control, the culture medium containing virus particles that were capable of inhibiting TSPYL5 expression, prepared by the same manner as described in Example <6-l>, was used. The cells were cultured in a 37^°C , 5% C0₂ incubator for 10 days, followed by the investigation of sphere formation.

Western blotting was performed by the same manner as described in Example <1-1> using the primary antibodies of ALDH1A1, ALDH1A3 (Abeam), CD133 (Biorbyt) , Oct3/4 (Millipore) , CD44, Sox2, Nanog, and β-actin (Cell Signaling) , which have been used as the cancer stem cell markers .

As a result, as shown in Figure 6A, the size of sphere, the major characteristics of tumorigenic transformation, was comparatively small when TSPYL5 expression was inhibited in the lung cancer cell line, and the expressions of major cancer stem cell markers were reduced as well, indicating that the cancer stem cell growth was suppressed (Figure 6A) . <6-3> Investigation of sphere formation according to the over-expression of TSPYL5 in the lung cancer cell line

The present inventors investigated sphere formation according to the over-expression of TSPYL5.

Particularly, 2xl0⁴ H460 cells were suspended in DMEM (Invitrogen) containing stem cell-permissive medium. DMEM- 12 (Invitrogen) was mixed with 20 ng/ml EGF, 20 ng/ml basic fibroblast growth factor (bFGf ) , and B27 serum- free supplement (50x; Invitrogen) , which was loaded in 60 mm plate pre-coated with 0.8% agarose. Then, the suspended cells above were loaded in the plate for culture. As for the control, the culture medium containing virus particles that were capable of inhibiting TSPYL5 expression, prepared by the same manner as described in Example <6-l>, was used. The cells were cultured in a 37^°C , 5% C0₂ incubator for 10 days, followed by the investigation of sphere formation.

Particularly, 2xl0⁴ H460 cells and the control H460 with TSPYL5 over-expressed according to the same manner as described in Example <4-l> were suspended in DMEM (Invitrogen) containing stem cell-permissive medium, followed by the investigation of sphere formation by the same manner as described in Example <6-2>.

Western blotting was also performed by the same manner as described in Example <1-1>. As a result, as shown in Figure 6B, the size of sphere, the major characteristics of tumorigenic transformation, was increased when TSPYL5 was over-expressed in the lung cancer cell line H460, and the expressions of major cancer stem cell markers were increased as well, indicating that the cancer stem cell growth was increased (Figure 6B) .

Therefore, it was confirmed that the inhibition of TSPYL5 resulted in the suppression of the tumorigenic cell growth .

Example 7: Investigation of TSPYL5 expression over the regulation of the cancer stem cell marker ALDHl

The present inventors investigated whether or not the regulation of the cancer stem cell marker ALDHl expression could affect the expression of TSPYL5.

Particularly, to inhibit the expressions of ALDH1A1 and ALDH1A3 genes in A549 cells which demonstrated high expressions of ALDHlAl and ALDHlA3 proteins, stealth-RNAi [ALDH1A1 forward primer: 5¹ -GAGAGUACGGUUUCCAUGA- 3¹ (SEQ. ID. NO: 26), reverse primer: 5 ' -UCAUGGAAACCGUACUCUC-3 ' (SEQ . ID. NO: 27); ALDHlA3 forward primer: 5¹ -CACAGAUGACAACGUCGUA-3¹ (SEQ. ID. NO: 28), reverse primer: 5 ' -UACGACGUUGUCAUCUGUG- 3' (SEQ. ID. NO: 29), Bioneer] was used. To insert the siRNA into the cell, 100 nM of RNAi was treated to 2x10^s A549 cells, and the cells were reacted in penicillin- streptomycin solution (Hyclone) free medium for 4 - 6 hours in the presence of lipofectamine RNAi MAX (Invitrogen) . For the negative control (siControl) , Scrambled StealthTM RNA molecule was used. Then, the medium was replaced with the medium containing penicillin-streptomycin, followed by culture for 72 hours.

In the meantime, to over-express ALDH1A1 and ALDH1A3 genes in H460 cells, ALDH1A1 and ALDH1A3 over-expressing clones were constructed and inserted in H460 cells. Amplification was performed using the forward primers [ALDH1A1 forward primer: 5 ' -ATATAAGCTTATGTCATCCTCAGGCACGCC- 3¹ (SEQ. ID. NO: 30), ALDH1A3 forward primer: 5'- ATATAAGCTTATGGCCACCGCTAACGGGGC-3¹ (SEQ. ID. NO: 32)] and the reverse primers [ALDH1A1 reverse primer: 5'- ATATGAATTCTTATGAGTTCTTCTGAGAGATTTTC-3¹ (SEQ. ID. NO: 31), ALDH1A3 reverse primer: 5¹ -ATATGAATTCTCAGGGGTTCTTGTCGCCAAG- 3' (SEQ. ID. NO: 33)] designed to contain Hindlll restriction enzyme recognition site at 5 ' -end and EcoRI restriction enzyme recognition site at 3 ' -end as follows: predenaturation at 94 ^°C for 5 minutes, denaturation at 94 ^°C for 1 minute, annealing at 56 ^"C for 1 minute, polymerization at 72 ^°C for 1.5 minute, 30 cycles from denaturation to polymerization, and final extension at 72 ^°C for 5 minutes. As a result, ALDH1A1 and ALDH1A3 genes were obtained. The PCR product obtained above and pcDNA3.1 vector (Invitrogen, USA) were treated with restriction enzymes and ligated by ligase, resulting in the construction of fibulin-3 over-expressing pcDNA3.1/ALDHlAl and ALDH1A3 vectors . 2 ug of the said ALDH1A1 or ALDH1A3 over-expressing vector was introduced in 2xl0⁵/ml of H460 cells by using lipofectamine 2000 in penicillin- streptomycin free medium, followed by reaction for 4 - 6 hours. Then, the medium was replaced with the fresh medium supplemented with 100 units/ml penicillin-streptomycin, followed by culture for 48 hours.

<7-l> TSPYL5 expression according to the inhibition of

ALDH1A and ALDH1A3

For the further experiment, the present inventors first inhibited the expressions of ALDH1A1 and ALDH1A3 genes in A549 cells.

Western blotting was performed by the same manner as described in Example <1-1> using the primary antibodies of

ALDH1A1 , ALDH1A3 (Abeam), TSPYL5, PTEN, β-catenin, Jaggedl, Notchl, Notch2, Notch3 (Santa Cruz), Oct4 (Millipore) ,

Cdl33 (Biorbyt), CD44, Sox2, Nanog, and β-actin (Cell

Signaling) , which have been used as the cancer stem cell markers .

As a result, as shown in Figure 7A, when ALDH1A1 and ALDH1A3 expressions were inhibited, the expressions of Sox2 , Oct4, and β-catenin were reduced, but the expressions of CD44, Cdl33, Nanog, Notch, and TSPYL5 were not changed, suggesting that TSPYL5 regulated ALDH1 at a higher level (Figure 7A) .

Colony formation assay was performed with A549 and another A549 with ALDH1A1 and ALDH1A3 inhibited by the same manner as described in Example <3-2>.

As a result, as shown in Figure 7B, when ALDH1A1 and ALDH1A3 expressions were inhibited, the cell growth was significantly inhibited (Figure 7B) .

<7-2> TSPYL5 expression according to the over-expression of ALDH1A and ALDH1A3

For the further experiment, the present inventors induced the over-expression of ALDH1A1 and ALDH1A3 genes in H460 cells.

Western blotting was performed by the same manner as described in Example <1-1> using the primary antibodies of ALDH1A1, ALDH1A3 (Abeam), TSPYL5, PTEN, β-catenin, Jaggedl, Notchl, Notch2, Notch3 (Santa Cruz), Oct3/4 (Millipore) , Cdl33 (Biorbyt), CD44, Sox2, Nanog, and β-actin (Cell Signaling) , which have been used as the cancer stem cell markers .

As a result, as shown in Figure 7C, when ALDH1A1 and ALDH1A3 were over-expressed, the expressions of Sox2 , Oct4 , and β-catenin were increased, but the expression of TSPYL5 was not changed (Figure 7C) .

Colony formation assay was performed with H460 and another H460 with ALDHlAl and ALDH1A3 over-expressed by the same manner as described in Example <3-2>.

As a result, as shown in Figure 7D, when ALDHlAl and ALDH1A3 were over-expressed, the cell growth was significantly increased (Figure 7D) . Example 8 : Investigation of metastasis according to the inhibition of ALDHl

The present inventors performed Western blotting and the experiment using matrigel kit with the lung cancer cell line A549 with ALDHlAl and ALDHlA3 inhibited and H462 with ALDHlAl and ALDH1A3 over-expressed, in order to investigate how the cell migration/invasion was affected by the regulation of ALDHlAl and ALDH1A3 genes.

Particularly, the migration/invasion assay was performed with A549, A549 with ALDHlAl and ALDHlA3 inhibited, the control H460, and H460 with ALDHlAl and

ALDH1A3 over-expressed by the same manner as described in

Example 2. Western blotting was also performed by the same manner as described in Example <1-1>.

As a result, as shown in Figures 8A and 8C, metastasis was suppressed by the inhibition of ALDHlAl and ALDHlA3 expressions. The expression of E-cadherin, the epithelial cell marker, one of EMT markers, was increased, while the expressions of N-cadherin, Vimentin, Twist, Snail, and Slug, the mesenchymal cell markers, were inhibited. Cell migration/invasion was significantly inhibited in A549 cells with ALDH1A1 and ALDH1A3 inhibited (Figure 8A) . In the meantime, the expressions of N-cadherin, Vimentin, Twist, Snail, and Slug, the mesenchymal cell markers, were increased but the expression of E-cadherin, the epithelial cell marker, was reduced in H460 with TSPYL5 over-expressed (Figure 8C) .

The present inventors performed the migration/invasion assay using transwell (Falcon, USA) by the same manner as described in Example 2, in order to measure the cancer cell metastasis.

Particularly, A549, A549 with ADH1A1 and ALDH1A3 inhibited, the control H460, and H460 with ALDH1A1 and ALDH1A3 over-expressed were cultured, which were distributed in the upper chamber of transwell (5xl0⁴ cells) . The lower chamber was filled with 500 ul of RPMI 1640 supplemented with 7% FBS . Another transwell chamber was pre-treated with 100 ul of matrigel (20 ug/well) for coating. The cells were maintained in a 37 ^°C , 5% C0₂ incubator for 40 hours and then the membrane in the upper chamber was stained with crystal violet, followed by observation under microscope to count the number of stained cells.

As a result, as shown in Figures 8B and 8D, the migration/invasion capacity was reduced by the inhibition of ALDHlAl and ALDH1A3 genes, while the migration/invasion of cell was increased by the over-expression of ALDHlAl and ALDH1A3 genes (Figures 8B and 8D) .

Therefore, it was confirmed that the expressions of EMT markers and the migration/invasion capacity could be changed by the regulation of ALDHlAl and ALDH1A3 genes.

Example 9 : Inhibition of sphere formation in various cancer cells by regulating the expression of TSPYL5

The present inventors inhibited the expression of TSPYL5 in Calu3 (adenocarcinoma cell line) , HepG2 (liver cancer cell line) , and Panel (pancreatic cancer cell line) that express as many TSPLY5 gene as the non-small cell lung cancer cell line A549 by the same manner as described in Example <6-2> and then investigated sphere formation therein.

Particularly, 2xl0⁴ cells of each cell line were suspended in DMEM (Invitrogen) containing stem cell- permissive medium. DMEM-12 (Invitrogen) was mixed with 20 ng/ml EGF, 20 ng/ml basic fibroblast growth factor (bFGf) , and B27 serum- free supplement (50x; Invitrogen) , which was loaded in 60 mm plate pre-coated with 0.8% agarose. Then, the suspended cells above were loaded in the plate for culture. As for the control, the culture medium containing virus particles that were capable of inhibiting TSPYL5 expression, prepared by the same manner as described in Example <6-l>, was used. The cells were cultured in a 37^°C , 5% C0₂ incubator for 10 days, followed by the investigation of sphere formation.

Western blotting was also performed using the primary antibodies used as the cancer stem cell markers ALDH1A1 or ALDH1A3 and β-actin (Cell Signaling) by the same manner as described in Example <1-1>.

As a result, as shown in Figure 9, the size of sphere, the major characteristics of tumorigenic transformation, was comparatively small when TSPYL5 expression was inhibited in Calu3 (adenocarcinoma cell line) , HepG2 (liver cancer cell line) , and Panel (pancreatic cancer cell line) , and the expression of the cancer stem cell marker ALDH1 was reduced, just like in the lung cancer cell line A549, indicating that the cancer stem cell growth was suppressed (Figure 9) .

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims .

Claims

[CLAIMS ]

[Claim l]

A pharmaceutical composition for preventing and inhibiting cancer metastasis comprising the TSPYL5 (testis- specific protein, Y-encoded-like 5) expression or activity- inhibitor as an active ingredient.

[Claim 2]

The pharmaceutical composition for preventing and inhibiting cancer metastasis according to claim 1, wherein the TSPYL5 expression inhibitor is the antisense nucleotide, small interfering RNA, or shRNA (short hairpin RNA) that binds complementarily to TSPYL5 mRNA. [Claim 3]

The pharmaceutical composition for preventing and inhibiting cancer metastasis according to claim 1, wherein the TSPYL5 activity inhibitor is selected from the group consisting of the compounds, peptides, peptide mimetics, and antibodies which are complementarily binding to TSPYL5 protein.

[Claim, 4]

The pharmaceutical composition for preventing and inhibiting cancer metastasis according to claim 1, wherein the TSPYL5 expression or activity inhibitor characteristically inhibits the growth of cancer stem cell and the metastasis thereof. [Claim 5]

The pharmaceutical composition for preventing and inhibiting cancer metastasis according to claim 1, wherein the cancer is selected from the group consisting of skin cancer, breast cancer, uterine cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, and stomach cancer.

[Claim 6]

A kit for inhibiting cancer stem cell growth comprising the TSPYL5 expression or activity inhibitor.

[Claim 7]

A method for inhibiting cancer stem cell growth containing the step of treating the TSPYL5 expression or activity inhibitor to cancer cells.

[Claim 8]

A method for screening a cancer metastasis inhibitor candidate comprising the following steps:

1) preparing the cell line expressing TSPYL5; 2) treating the sample material to the cell line of step 1) ;

3) measuring the expression or activity of ALDH (aldehyde dehydrogenase) in the cell line; and

4) selecting the sample material that could inhibit the expression or activity of ALDH, compared with the expression or activity thereof in the control not-treated with the sample material. [Claim 9]

A method for screening a cancer stem cell growth inhibitor candidate comprising the following steps:

1) preparing the cell line expressing TSPYL5;

2) treating the sample material to the cell line of step 1) ;

3) measuring the expression or activity of ALDH in the cell line; and

[Claim 10]

The screening method according to claim 8 or claim 9, wherein the cell line expressing TSPYL5 of step 1) is prepared by irradiating cancer cells. iClaim ll]

The screening method according to claim 10, wherein the irradiation is either single irradiation with 2 Gy or 4 Gy or the fractionated irradiation accomplished with 2 Gy by 2 ~ 5 divided times.

[Claim 12]

A method for preventing cancer metastasis containing the step of administering a pharmaceutically effective dose of the TSPYL5 expression or activity inhibitor to a subject having cancer. [Claim 13]

A method for inhibiting cancer metastasis containing the step of administering a pharmaceutically effective dose of the TSPYL5 expression or activity inhibitor to a subject having cancer.

[Claim 14]

A use of the TSPYL5 expression or activity inhibitor for a pharmaceutical composition for preventing and inhibiting cancer metastasis. [Claim 15]

A use of the kit for inhibiting cancer stem cell growth comprising the TSPYL5 expression or activity- inhibitor .