KR20240153453A - Pharmaceutical composition for preventing or treating cancer comprising a shRNA for inhibiting IGFBP5 expression as an active ingredient - Google Patents

Abstract

The IGFBP5 expression inhibitor and/or IGFBP5 mutant protein of the present invention has a strong inhibitory effect on the metastasis, invasiveness, and selective tumorigenesis of cancer cells, and thus can be usefully used in the treatment of various diseases caused by abnormal growth of cells, especially degenerative diseases such as primary and metastatic solid cancers and leukemia.

Description

{Pharmaceutical composition for preventing or treating cancer comprising a shRNA for inhibiting IGFBP5 expression as an active ingredient}

The present invention confirms that the tumor protein kinase enzyme PLK1 induces tumor formation and cancer metastasis through IGFBP5 phosphorylation, and provides shRNA and the like that are effective in suppressing the expression of IGFBP5, thereby reducing tumor formation and cancer mobility and invasiveness through suppression of the expression of IGFBP5.

IGFBP5 is known to bind to IGF-I and regulate IGF-1 signaling (Firth SM et al., Endocr Rev, (2002) 23(2002), pp.824-854). IGF-1 is an insulin-like growth factor important for the growth, development, and maintenance of tissues (Sahitya K Denduluri et al., Genes and Disease, 2 (2015), pp. 13-25). IGFBP5, which is found in all vertebrates, is located on human chromosome 2, is expressed in various human tissues, and encodes a protein of approximately 29 KDa. IGFBP5 consists of an N-terminal domain, an L-domain, and a C-terminal domain, and has a signal peptide that causes secretion into the extracellular matrix (ECM) and a nuclear localization sequence (NLS) that causes translocation into the nucleus (Mustafa Akkiprik et al., Breast Cancer Res, 212 (2008)). Both the N-terminal and C-terminal domains of IGFBP5 contain IGF-1 binding sites, and the C-terminal domain contains a binding site for ECM proteins (Hodgkinson SC et al., J Mol Endocrinol, 13 (1994), pp. 105-112). IGFBP5 is known to have a potential role in oncogenesis through two pathways, IGF-1-dependent and IGF-1-independent pathways (Mustafa Akkiprik et al., BMC cancer, 103 (2009)). IGFBP5 binds to IGF-1, prolongs the half-life of IGF-1, and may act as a reservoir for release when needed in tissues (Mohan S et al., J Endocrinol, 175 (2002), pp. 19-31). IGFBP-5 binds to both the cell surface and the extracellular matrix (ECM), and binding of IGFBP-5 to the ECM or heparin decreases the affinity of IGFBP-5 for IGF-I by 8- to 17-fold and enhances the biological action of IGF-I on cells (Jones JI et al., J Cell Biol, 121 (1993), pp.679-87.). In the IGF-I-independent pathway, IGFBP5 can enter the cytoplasm through binding to a putative receptor. Cytoplasmic accumulation of IGFBP5 by interaction with other proteins exhibits anti-apoptotic effects and stimulates metastasis, whereas IGFBP5 that enters the nucleus through binding to Importin-β enhances apoptosis. IGFBP5 mRNA was detected in breast cancer tissue specimens, and higher expression was observed in tumor specimens compared with adjacent normal tissues. Additionally, it has been observed that IGFBP5 protein is localized in the cytoplasm (Mustafa Akkiprik et al., Breast Cancer Res, 212 (2008)).

Expression of IGFBP5 has been linked to breast cancer (Mustafa Akkiprik et al., Breast Cancer Res, 212 (2008)), colorectal cancer (Yu Deng et al., International Journal of General Medicine, 15 (2022), pp.6485-6497), pancreatic cancer (Sarah K et al., Molecular Carcinogenesis, 45 (2006), pp.814-827), ovarian cancer (Graeme Walker et al., Clinical Cancer Research, 13 (2007), pp.1438-1444), glioblastoma (Vani Santosh et al., Cancer Epidemiology Biomarkers & Prevention, 19 (2010), pp.1399-1408), and non-functioning pituitary adenoma (NEPA) (Francoise Galland, Endocrine-Related Cancer, 17 (2010), It is observed in malignant tumors including (pp.361-371).

In clinical data of breast cancer, the expression of IGFBP5 was higher in T1 breast carcinoma than in benign breast epithelium, and higher in T1N1 than in T1NO carcinoma. It has been reported that IGFBP5 can be one of the markers predicting lymph node metastasis in T1 invasive breast, which suggests that IGFBP5 expression is associated with metastasis (Huamin Wang MD et al., The Breast journal, 14 (2008), pp.261-267), but its role in mediating EMT is not precisely known.

Polo-like kinase 1 (PLK1) is well known as a protein kinase enzyme that causes carcinogenesis (Barr et al., Nat Rev Mol Cell Biol 5 (2004) 429-440; Yim and Erikson, Mutation Research Reviews Mutation Research, 761 (2014) 31-39), and recent studies have revealed that it also functions as a kinase enzyme that induces metastasis (Cai et al., Am J Transl Res 8 (2016) 4172-4183; Wu et al., eLife 5 (2016) e10734; Shin et al., Oncogene, 39 (2020), pp.767-785), and it is being studied as a target molecule for cancer treatment. Based on this, research on the development of anticancer drugs through the development of PLK1 inhibitors is being competitively conducted mainly by multinational companies (J. Van den Bossche, 36 (2016), pp.749-786; Rosie Elizabeth Ann Gutteridge, Molecular Cancer Therapeutics, 15 (2016), pp.1427-1435; Yim, Anti-Cancer Drugs 24(2013) 999-1006; Yim and Erikson, Mutation Research Reviews Mutation Research, 761 (2014) 31-39). PLK1 has a kinase activation domain with kinase activity and a polo box domain that binds substrates, and phosphorylation at position T210 induces PLK1 activation. Activated PLK1 kinase is an enzyme that phosphorylates Ser/Thr residues of substrates (Barr et al., Nat Rev Mol Cell Biol 5 (2004) 429-440). Functionally, its expression is increased in growing and dividing cells, and its expression and activity peak during the cell division phase of the cell cycle. Therefore, it is also known to have a high expression rate in rapidly growing cancer cells (Yim, Anti-Cancer Drugs 24(2013) 999-1006; Yim and Erikson, Mutation Research Reviews Mutation Research, 761 (2014) 31-39), and it has been experimentally reported that its expression increases in many cases during the stage-by-stage malignancy process of cancer cells. In particular, it has been reported that its expression increases as the malignancy stage increases in non-small cell lung cancer, head and neck cancer, laryngeal cancer, breast cancer, liver cancer, endometrial cancer, colon cancer, ovarian cancer, pancreatic cancer, and prostate cancer (Yim, Anti-Cancer Drugs 24(2013) 999-1006; Yim and Erikson, Mutation Research Reviews Mutation Research, 761 (2014) 31-39). Recent research reports and our own studies have shown that activated PLK1 is involved in increasing cancer metastasis (Cai et al., Am J Transl Res 8 (2016) 4172-4183; Wu et al., eLife 5 (2016) e10734; Shin et al., Oncogene, 39 (2020), pp.767-785), and thus the value of PLK1 as a molecular target for treating metastatic cancer has recently begun to be highlighted. Therefore, it is expected that anticancer agents targeting the regulation of the function of PLK1 phosphorylation substrates will be effective in the treatment of not only primary cancer but also metastatic cancer.

Although metastatic cancer shows different differences depending on the cancer type, it can account for up to 90% of all cancer patient deaths, so its risk can be estimated (Valastyan, S. and Weinberg, R.A., Cell 147 (2011) 275-292; Khan, I. and Steeg, P.S., Lab Invest 98 (2018) 198-210). It is known that even if the primary cancer is treated in chemotherapy, the survival rate is greatly reduced due to cancer recurrence, etc. if metastasis cannot be prevented (Valastyan, S. and Weinberg, R.A., Cell 147 (2011) 275-292; Shibue, T. and Weinberg, Semin Cancer Biol 21 (2011) 99-106). In cancer treatment, the inhibition of cancer cell proliferation and the inhibition of metastasis do not always appear at the same time. Therefore, the development of a treatment that can suppress cancer metastasis can dramatically improve the treatment efficiency of cancer by increasing the survival rate of many cancer patients. Therefore, the development of a treatment target or treatment agent that effectively suppresses cancer metastasis and invasion for the treatment of metastatic cancer is necessary.

Valastyan, S. and Weinberg, R.A., Cell 147 (2011) 275-292; Shibue, T. and Weinberg, Semin Cancer Biol 21 (2011) 99-106

The present invention has been completed by confirming a decrease in metastasis, invasiveness, and selective tumorigenesis of cancer cells by suppressing the expression of IGFBP5, and provides a pharmaceutical composition for preventing or treating cancer containing shRNA effective in suppressing the expression of IGFBP5.

In addition, the present invention aims to provide a pharmaceutical composition for preventing or treating cancer, which comprises as an active ingredient an IGFBP5 mutant protein in which a region phosphorylated by PLK1 is mutated.

In addition, the present invention aims to provide a pharmaceutical composition for preventing or treating cancer, comprising the IGFBP5 shRNA and IGFBP mutant protein as active ingredients.

The pharmaceutical composition of the present invention described above inhibits metastasis in solid cancers derived from various tissues and exhibits potent inhibitory activity against metastasis, invasiveness and selective tumorigenesis of cancer cells appearing in non-small cell lung cancer cells, so that it can effectively prevent and treat various solid cancers and metastatic cancers.

However, the technical problems to be achieved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

Wild-type IGFBP5 (insulin like growth factor binding protein 5) is a protein consisting of the amino acid sequence of sequence number 1 and encoded by the base sequence of sequence number 5.

To solve the above problem, the present invention provides a pharmaceutical composition for preventing or treating cancer containing an IGFBP5 expression inhibitor as an active ingredient.

As one embodiment of the present invention, the IGFBP5 expression inhibitor may be an aptamer, siRNA, shRNA, and miRNA comprising an oligonucleotide complementary to a portion of IGFBP5 mRNA.

As another embodiment of the present invention, the IGFBP5 expression inhibitor may be an oligonucleotide comprising a base sequence complementary to the base sequence of SEQ ID NO: 11, SEQ ID NO: 15, or SEQ ID NO: 19.

As another embodiment of the present invention, the IGFBP5 expression inhibitor may be an shRNA comprising or consisting of any one of the base sequences of SEQ ID NO: 6 to SEQ ID NO: 8, but preferably, it may be an shRNA comprising or consisting of the base sequence of SEQ ID NO: 6.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer containing the shRNA as an active ingredient.

As one embodiment of the present invention, the composition may additionally include an IGFBP5 mutant protein in which amino acid residues 256 and/or 269 of IGFBP5 of SEQ ID NO: 1 are substituted for Threonine with Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Methionine or Tryptophan.

As another embodiment of the present invention, the mutant protein may comprise or consist of the amino acid sequence of SEQ ID NO: 2.

As another embodiment of the present invention, the mutant protein may comprise or consist of the amino acid sequence of SEQ ID NO: 3.

As another embodiment of the present invention, the 256th amino acid residue of SEQ ID NO: 3 may be alanine.

As another embodiment of the present invention, the IGFBP5 mutant protein may comprise or consist of the amino acid sequence of SEQ ID NO: 4.

In another embodiment of the present invention, the 269th amino acid of sequence number 4 may be alanine.

As another embodiment of the present invention, the composition may inhibit cancer metastasis by reducing the mobility and invasiveness of cancer cells.

In another embodiment of the present invention, the cancer may be a solid cancer, and the solid cancer may be at least one cancer selected from the group consisting of glioblastoma, ovarian cancer, bladder cancer, non-functioning pituitary adenoma, esophageal cancer, renal cancer, breast cancer, stomach cancer, rectal cancer, cervical cancer, endometrial cancer, lung cancer, and pancreatic cancer, and the lung cancer may be non-small cell lung cancer, and the non-small cell lung cancer may particularly be adenocarcinoma.

In addition, the present invention provides a method for preventing or treating cancer, comprising a step of administering the IGFBP5 expression inhibitor and/or the IGFBP5 mutant to a subject.

In one embodiment of the present invention, the subject may be a human being in need of prevention or treatment of cancer, a patient who has already developed cancer and is undergoing treatment, or a patient in need of prevention of metastasis after complete cure of cancer. In addition, the present invention provides a use of the IGFBP5 expression inhibitor for the manufacture of a medicament for the prevention or treatment of cancer.

The IGFBP5 expression inhibitor and/or IGFBP5 mutant protein of the present invention has a strong inhibitory effect on the metastasis, invasiveness, and selective tumorigenesis of cancer cells, and thus can be usefully used in the treatment of various diseases caused by abnormal growth of cells, especially degenerative diseases such as primary and metastatic solid cancers and leukemia.

Figures 1a and 1b show the results of analyzing the survival rate according to the expression of PLK1 and IGFBP5 in a clinical analysis targeting patients with adenocarcinoma.
Figure 1a: Results of analysis of overall survival rates according to PLK1 and IGFBP5 expression in patients with lung adenocarcinoma by stage using KM plotter.
Figure 1b: Results of analyzing the increase in PLK1 and IGFBP5 expression in patients with lung adenocarcinoma by stage using heat map analysis.
Figures 2a to 2g show the results of observing the expression level of IGFBP5 during the mesenchymal transition process induced by TGF-β.
Figure 2a: The results of immunoblotting to observe IGFBP5 expression when EMT occurred after treating A549 cells and H358 cells with 5 ng/ml TGF-β. To confirm whether EMT was well induced, the expression of epithelial marker E-cadherin and mesenchymal markers N-cadherin, Vimentin, and SNAI1 was also observed.
Figure 2b: A graph numerically expressing the immunoblot data observing IGFBP5 expression when EMT occurred after treatment with 5 ng/ml TGF-β in A549 and H358 cells.
Figure 2c: The results of observing the IGFBP5 mRNA level using real-time polymerase chain reaction (Real-time PCR) when EMT occurred after treating A549 cells and H358 cells with 5 ng/ml TGF-β.
Figure 2d: Results of analyzing the expression of IGFBP5, PLK1, and p-PLK1 in lung fibroblasts MRC5, primary lung cancer A549, and metastatic lung cancer H460, H358, and H1299 using immunoblotting.
Figure 2e: Results of analyzing the expression of IGFBP5, PLK1, and p-PLK1 in lung fibroblasts MRC5, primary lung cancer A549, and lung adenocarcinoma stage-specific cell lines (H522, H1373, H2009) using immunoblotting.
Figure 2f: The results of immunoblotting to observe IGFBP5 expression when EMT occurred after treating A549 cells and H358 cells with 5 ng/ml TGF-β. To confirm whether EMT was well induced, the expression of epithelial marker E-cadherin and mesenchymal markers N-cadherin, Vimentin, and SNAI1 was also observed.
Figure 2g: This is a graph numerically expressing the immunoblot data observing IGFBP5 expression when EMT occurred after treatment with 5 ng/ml TGF-β in A549 and H358 cells.
Figure 3 shows the results of analyzing the survival rate of solid cancer patients according to the expression of IGFBP5 in clinical data according to an embodiment of the present invention.
A: This is a graph showing the results of analyzing the survival rate according to IGFBP5 expression in patients with cervical cancer (left graph), endometrial cancer (middle graph), and breast cancer (right graph) using KM PLOTTER analysis.
B: This is a graph showing the results of analyzing the survival rate according to IGFBP5 expression in patients with cervical bladder cancer (left graph), esophageal cancer (middle graph), and ovarian cancer (right graph) using KM PLOTTER analysis.
C: This is a graph showing the results of analyzing the survival rate according to IGFBP5 expression in patients with renal cell carcinoma (left graph) and gastric cancer (middle graph) using KM PLOTTER analysis.
Figure 4 shows the results of analyzing the interaction between PLK1 and IGFBP5 and the phosphorylation and phosphorylation sites of IGFBP5 by the PLK1 activated form.
A: This is the result of observing the interaction between IGFBP5 and PLK1 by performing immunoprecipitation using PLK1 antibody under conditions of treating A549 cells with 5 ng/ml TGF-β.
B: This is the result of observing the interaction between IGFBP5 and PLK1 by performing immunoprecipitation using PLK1 antibody under conditions of treating H460 cells with 2.5 ng/ml TGF-β.
C: This is the result of cloning IGFBP5 into the pGEX-4T-1 vector tagged with GST to purify GST-labeled IGFBP5.
D: The result of purifying GST-tagged IGFBP5 using GST purification.
E: Results of performing a phosphorylation enzyme reaction with active PLK1 (PLK1 TD) using GST-labeled IGFBP5.
Figures 5a to 5d show the results of expressing non-phosphorylated point mutants to find the site of IGFBP5 phosphorylated by PLK1.
Figure 5a: Results of confirming the phosphorylation potential candidate site of IGFBP5 phosphorylated by PLK1 using liquid chromatography quantitative analysis.
Figure 5b: The result of substituting the candidate site of IGFBP5 with alanine, a non-phosphorylated form, using the partial-specific mutagenesis method before performing the phosphorylation enzyme reaction.
Figure 5c: The results of performing a phosphatase reaction using a non-phosphorylated point mutant protein in which the phosphorylation candidate site of IGFBP5 was substituted with alanine using a partial-specific mutagenesis method and active PLK1.
Figure 5d: It was confirmed that threonine at position 265 and serine at position 269 are conserved in higher animals including humans.
Figure 6 shows the results of observing the expression of EMT markers after expressing RFP-IGFBP5 via the Tet-On 3G inducible system using lentivirus.
A: This is the result of cloning IGFBP5 into the pLVX-TRE3G-eRFP vector to create lentivirus.
B: This is a diagram illustrating the Tet-On 3G inducible system, a system for RFP-IGFBP5 expression.
C: This is the result of observing the presence or absence of IGFBP5 protein overexpression and changes in epithelial marker (E-cadherin) and mesenchymal transition marker (Vimentin) by immunoblotting after expressing phosphorylated and non-phosphorylated point mutants of threonine 265 of IGFBP5 in lung cancer cell A549.
D: This is the result of observing the presence or absence of IGFBP5 protein overexpression and changes in mesenchymal transition markers (Vimentin, N-cadherin) by immunoblotting after expressing phosphorylated and non-phosphorylated point mutants of serine 269 of IGFBP5 in lung cancer cells A549.
Figure 7 is an experiment measuring the effect of overexpression of phosphorylated and non-phosphorylated point mutants of IGFBP5 on the motility and invasiveness of cancer cells in lung cancer cells A549.
A: This is the result of observing the presence or absence of RFP-IGFBP5 mRNA overexpression and changes in epithelial marker (CDH1) and mesenchymal transition marker (CDH2, VIM, SNAI1) mRNA by chain reaction (real-time PCR) after expressing IGFBP5 phosphorylated and non-phosphorylated point mutants in lung cancer cells A549.
B: This is the result of observing the cell migration pattern through a migration assay using an insert in lung cancer cell A549 expressing IGFBP5 phosphorylated and non-phosphorylated point mutants.
C: This is the result of observing the invasiveness of cells through an invasion assay using matrigel and an insert in lung cancer cells A549 expressing phosphorylated and non-phosphorylated point mutants of IGFBP5.
D: This is a graph analyzing caspase-3 activity in total cancer cells expressing the original or phosphorylated point mutants of IGFBP5 using the caspase-3 assay.
Figure 8 shows the results of confirming the effect of IGFBP5 shRNA treatment, a substance that suppresses IGFBP5 mRNA expression under metastatic conditions, on the metastatic inhibition of cancer cells and the effect on cell survival.
A: This is a graph measuring the degree of IGFBP5 expression inhibition by IGFBP5 shRNA according to the target sequence using real-time polymerase chain reaction (real-time PCR).
B: Results of immunoblotting to observe changes in epithelial-mesenchymal transition markers at the protein level by shRNA treatment of IGFBP5 in a TGF-β-induced metastatic environment.
C: This is a graph numerically expressing immunoblot data observing changes in epithelial-mesenchymal transition markers at the protein level by shRNA treatment of IGFBP5 in a TGF-β-induced metastatic environment.
D: TGF- The results were observed by observing the change in the mRNA level of epithelial-mesenchymal transition markers by shRNA treatment of IGFBP5 in an inducible metastatic environment using real-time polymerase chain reaction (real-time PCR).
Figure 9 shows the results of observing the inhibitory effect on the mobility and invasiveness of cancer cells and the change in survival rate by shRNA treatment of IGFBP5, an IGFBP5 mRNA expression inhibitor, in a metastatic environment induced by TGF-β of IGFBP5. In addition, the results of observing the mobility and invasiveness of cancer cells by IGFBP5 phosphorylation point mutants.
A: This is the result of observing the effect of suppressing cell mobility by shRNA treatment of IGFBP5 using Transwell in a metastatic environment induced by TGF-β.
B: The results of observing the effect of IGFBP5 shRNA treatment on the inhibition of cell invasiveness in a metastatic environment induced by TGF-β using Transwell and matrigel.
C: This is a graph analyzing the change in caspase-3 activity by shRNA treatment of IGFBP5 in a TGF-β-induced metastatic environment using the Caspase-3 assay.
D: The results of observing the cell mobility pattern through a mobility experiment using an insert in lung cancer cell A549 expressing IGFBP5 phosphorylated and non-phosphorylated point mutants.
E: The results of observing the invasiveness of cells through an invasiveness experiment using matrigel and an insert in lung cancer cells A549 expressing phosphorylated and non-phosphorylated point mutants of IGFBP5.
Figure 10 shows the results of observing the change in cancer cell survival rate by shRNA treatment of IGFBP5, a substance that suppresses IGFBP5 mRNA expression in solid cancers other than lung cancer.
A: This is a graph measuring the degree of IGFBP5 expression inhibition using real-time polymerase chain reaction (real-time PCR) after treating HeLa cells, a cervical cancer cell line, and BT-20 breast cancer cells with IGFBP5 shRNA.
B: This is a graph analyzing the change in caspase-3 activity by shRNA treatment of IGFBP5 in HeLa cells and BT-20 cells using the Caspase-3 assay.
C: This is a graph measuring IGFBP5 expression using real-time PCR after treating HeLa cells, a cervical cancer cell line, with IGFBP5 shRNA in a metastatic environment induced by TGF-β.
D: This is a graph analyzing the change in caspase-3 activity by shRNA treatment of IGFBP5 in a TGF-β-induced metastatic environment using HeLa cells using a Caspase-3 assay.

The present inventors, while studying PLK1 (Polo-like kinase 1), confirmed that phosphorylation of IGFBP5 by PLK1 contributes to cancer metastasis, and confirmed that when IGFBP5 expression is knocked down, cancer mobility and invasiveness are significantly reduced, thereby completing the present invention.

In addition, the present inventors explored the phosphorylation region of IGFBP5 by PLK1 and confirmed that inducing mutations therein resulted in a significant decrease in cancer mobility and invasiveness.

From the above, the present inventors provide a pharmaceutical composition for preventing or treating cancer containing an IGFBP5 inhibitor as an active ingredient.

In the present invention, the "IGFBP5 inhibitor" may be an agent that reduces IGFBP5 overexpressed in tumor tissues to a normal expression level, and more specifically, refers to an agent that reduces IGFBP5 phosphorylated by PLK1 to a level in normal tissues. Specifically, the IGFBP5 inhibitor may be an agent that reduces the expression level of IGFBP5 mRNA, and may be an IGFBP5 mutant that interacts with PLK1 as a competitive inhibitor of wild-type IGFBP5 but is not phosphorylated.

PLK1 is known to be overexpressed in various cancers, and research on the PLK1 molecule as a target for the development of cancer therapeutics is actively being conducted. However, PLK1 is a kinase involved in various signaling systems such as the cell cycle, so it is difficult to rule out the possibility that PLK1 inhibition may have other effects other than the effect of inhibiting tumor growth, and there is a problem that PLK1 does not perform its original function smoothly in normal cells.

There have been no reports on PLK1's interaction with IGFBP5.

In order to examine the clinical correlation between PLK1 and IGFBP5, the present inventors analyzed KM plotter data based on TCGA data to confirm the expression of PLK1 and IGFBP5 and the survival rate of patients in non-small cell lung cancer patients. As a result, we were able to find clinical significance in that the expression of PLK1 and IGFBP5 increases according to the cancer progression stage of non-small cell lung cancer patients and that this lowers the survival rate of patients. In addition, as a result of analyzing the number of patients with increased IGFBP5 expression in tumor tissues compared to normal tissues using a heat map, we were able to confirm that IGFBP5 expression was high in the patient group with a high stage (Example 1).

In addition, to confirm quantitative changes in IGFBP5 under conditions where metastasis occurred, the expression of IGFBP5 protein and mRNA was examined under conditions where TGF-β was treated, and it was observed that the expression of IGFBP5 increased together with the EMT marker (Example 2). From the above, it can be seen that the expression of IGFBP5 increases according to the progression of cancer and epithelial-mesenchymal transition.

Meanwhile, as a result of confirming whether solid cancers other than lung cancer show the same expression pattern of IGFBP5 as lung cancer, it was confirmed that in patients with cervical cancer, endometrial cancer, breast cancer, bladder cancer, esophageal cancer, ovarian cancer, kidney cancer, and stomach cancer, high expression of IGFBP5 was associated with a lower survival rate (Example 3).

Next, the inventors of the present invention aimed to target downstream molecules in the mechanism by which PLK1 is involved in cancer proliferation and metastasis. Specifically, to confirm whether the interaction between PLK1 and IGFBP5 is induced under conditions that induce metastatic cancer, they performed immunoprecipitation after inducing metastatic lung cancer with TGF-β and were able to observe increased binding of PLK1 and IGFBP5 (Example 4).

Meanwhile, IGFBP5 binds to IGF-I and regulates IGF-I signaling. The inventors of the present invention sought to develop a method that can block only the action at the stage of cancer occurrence and metastasis while maintaining the original function of IGFBP5 in normal cells. The inventors observed through clinical results that PLK1 and IGFBP5 were overexpressed in non-small cell lung cancer, especially in higher metastasis stages. Assuming that phosphorylation of IGFBP5 by PLK1 is a necessary condition for cancer metastasis, if only phosphorylation of IGFBP5 by PLK1 is prevented, cancer metastasis can be suppressed. Therefore, the inventors of the present invention searched for the phosphorylation region of IGFBP5 by PLK1 and selected Ser269 and Thr265 as candidate phosphorylation regions (Examples 4 and 5).

Next, the inventors of the present invention produced lentiviruses expressing mutants in which serine 269 and threonine 265 were substituted with alanine (non-phosphorylated form) or aspart (phosphorylated form), and transformed cancer cells using the lentiviruses, and confirmed the expression of EMT markers in cancer cells expressing each mutant. As a result, it was confirmed that the expression of epithelial markers increased and the expression of mesenchymal markers decreased in cancer cells expressing mutants in which serine 269 and threonine 265 were substituted with alanine, indicating that serine 269 and threonine 265 of IGFBP5 are effective as targets for blocking phosphorylation by PLK1 (Example 6).

More specifically, the inventors of the present invention conducted a cancer cell migration assay to observe the metastasis of cancer cells in A549 cells expressing phosphorylated and non-phosphorylated point mutants of IGFBP5 through specific experiments. As a result, it was observed that the cancer cell migration increased more in the experimental group expressing the phosphorylated point mutant of IGFBP5 than in the case of the positive control group treated with TGF-β (5 ng/ml). In contrast, the migration of cancer cells expressing the non-phosphorylated point mutant protein decreased (Example 7).

In addition, the inventors of the present invention conducted a specific experiment to investigate the effects of promoting and inhibiting the invasiveness of cancer cells using phosphorylated point mutants and non-phosphorylated point mutants of IGFBP5. The invasiveness of cancer cells was observed using an invasion assay using Matrigel. First, in order to evaluate the invasiveness, cells expressing each point mutant of IGFBP5 were dispensed into a Matrigel insert together with a medium without serum, and a medium containing serum was dispensed into an experimental plate and cultured for 5 days. The invaded cancer cells were stained with crystal violet and observed, and after dissolving in DMSO, the absorbance was measured at a wavelength of 590 nm. As a result of the study, it was observed that the lung cancer cell group expressing the phosphorylated point mutant protein of IGFBP5 had an increased invasiveness compared to the control group. In particular, high cancer cell invasiveness was observed in the phosphorylated point mutant protein of IGFBP5. On the other hand, the lung cancer cell group expressing the non-phosphorylated point mutant protein of IGFBP5 had a decreased invasiveness. Therefore, it was possible to observe the invasiveness-promoting effect of IGFBP5 phosphorylation point mutants in lung cancer cells and the invasiveness-inhibiting effect of non-phosphorylation point mutants in lung cancer cells (Example 7).

Next, the inventors of the present invention attempted to confirm whether cancer metastasis and invasion could be reduced by suppressing the expression of IGFBP5. To this end, shRNAs targeting the nucleotide sequences at positions 443-463 or 467-487 or 674-694 in the IGFBP5 mRNA sequence were designed and produced, and the IGFBP5 expression suppression efficacy of each was confirmed. As a result, all shRNAs suppressed the expression of IGFBP5, but in particular, high IGFBP5 expression suppression activity was confirmed in the shRNA targeting positions 443-463 of IGFBP5 mRNA (Example 8).

Using the shRNA that effectively suppresses the expression of the above IGFBP5, it was confirmed whether suppression of the expression of IGFBP5 could suppress the progression of epithelial-mesenchymal transition. As a result, it was confirmed that the progression of epithelial-mesenchymal transition was reduced in lung cancer cells treated with the above shRNA expression vector even in a metastasis-inducing environment by TGF-β (Example 9). In addition, it was confirmed that the above IGFBP5 shRNA significantly reduced the mobility and invasiveness of cancer cells even in a metastasis-inducing environment (Example 10).

Next, the inventors of the present invention confirmed whether IGFBP5 can be used as a target for the treatment of other solid cancers in which the expression of IGFBP5 is increased, and when the vector expressing the shRNA was treated in cervical cancer cells, it was confirmed that the vector expressing the shRNA promoted apoptosis of cancer cells, and this was confirmed to be the same in an environment in which metastasis was induced by TGF-β (Example 11).

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising an IGFBP5 expression inhibitor as an active ingredient.

In the present invention, the “IGFBP5 expression inhibitor” is an oligonucleotide containing a base sequence complementary to a part of IGFBP5 mRNA, and may be at least one selected from the group consisting of antisense oligonucleotides, aptamers, siRNA, shRNA, and miRNA that specifically bind to IGFBP5 mRNA.

In the present invention, the "antisense oligonucleotide" is DNA, RNA or a derivative thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA, which can bind to the complementary sequence in the mRNA and inhibit the translation of the mRNA into a protein. The antisense oligonucleotide sequence refers to a DNA or RNA sequence complementary to the IGFBP5 sequence of SEQ ID NO: 1 and capable of binding to the sequence. For example, it may contain or consist of a sequence complementary to any one sequence selected from the group consisting of the sequences of SEQ ID NOs: 3 and 4, but is not limited thereto. The length of the antisense oligonucleotide may be 5 to 100 bases, or 8 to 60 bases, or 10 to 40 bases, or 10 to 30 bases. The antisense oligonucleotide may be synthesized in vitro by a conventional method and administered in vivo, or may be administered in a form in which the antisense oligonucleotide is synthesized in vivo. The method for synthesizing an antisense oligonucleotide in a test tube can be synthesized by a biological/chemical method according to a conventional method in the technical field of the present invention. In addition, the form in which the antisense oligonucleotide is synthesized in a living body can be realized by using a recombinant vector expressing the antisense oligonucleotide, etc., and this method can be easily performed according to a conventional method in the technical field of the present invention. Therefore, the design of the antisense oligonucleotide of the present invention can be easily produced according to a method known in the technical field of the present invention with reference to the base sequence of SEQ ID NO: 1. For example, the antisense oligonucleotide may include a base sequence complementary to a base sequence selected from the group consisting of the sequences of SEQ ID NO: 3 or 4.

In the present invention, "aptamer" is a single-stranded oligonucleotide, and is about 20 to 60 nucleotides in size, and refers to a nucleic acid molecule having binding activity to a predetermined target. It has various three-dimensional structures depending on the sequence, and can have high affinity with a specific substance like an antigen-antibody reaction. The aptamer can inhibit the activity of a predetermined target by binding to the predetermined target. The aptamer of the present invention can be RNA, DNA, a modified nucleic acid, or a mixture thereof, and can also be in a linear or cyclic form. Preferably, the aptamer can bind to IGFBP5 mRNA and play a role in inhibiting the expression or activity of IGFBP5. Such an aptamer can be prepared by a person skilled in the art from the sequence of IGFBP5 of SEQ ID NO: 1 by a known method.

In the present invention, the terms "siRNA" and "shRNA" refer to nucleic acid molecules capable of mediating RNA interference or gene silencing, and are used as an efficient gene knockdown method or gene therapy method because they can suppress the expression of target genes. shRNA forms a hairpin structure by binding between complementary sequences within a single-stranded oligonucleotide, and in vivo, the shRNA is cleaved by dicer into small RNA fragments of 8 to 30 nucleotides in size, which become siRNA, a double-stranded oligonucleotide, and can specifically bind to mRNA having a complementary sequence to suppress its expression. Therefore, which means among shRNA and siRNA to use can be determined by the choice of a person skilled in the art, and if the mRNA sequences they target are the same, a similar expression reduction effect can be expected. For the purpose of the present invention, IGFBP5 can be suppressed by specifically acting on IGFBP5, cleaving the IGFBP5 mRNA molecule, and inducing RNA interference (RNAi, RNA interference) phenomenon. siRNA can be synthesized chemically or enzymatically. The method for producing siRNA is not particularly limited, and methods known in the art can be used. For example, a method of directly chemically synthesizing siRNA, a method of synthesizing siRNA using in vitro transcription, a method of cleaving long double-stranded RNA synthesized by in vitro transcription using an enzyme, an expression method through intracellular delivery of an shRNA expression plasmid or viral vector, and an expression method through intracellular delivery of a PCR (polymerase chain reaction)-induced siRNA expression cassette, but are not limited thereto.

In the present invention, "miRNA (micro RNA)" refers to a substance that naturally exists in cells and induces RNAi phenomenon and participates in the regulation of specific genes. Any miRNA capable of suppressing the expression or function of IGFBP5 of the present invention is included in the present invention without limitation to its type.

In addition, the present invention can provide an IGFBP5 mutant having S269 and/or T265 amino acids substituted for use in the prevention and treatment of cancer.

Meanwhile, PLK1 structurally contains an enzyme activity domain with kinase activity and a polo box domain that binds to a substrate, and thus, an IGFBP5 mutant in which amino acids are substituted at positions S269 and T265 maintains the binding affinity to PLK1. Accordingly, a mutant in which the phosphorylation domain amino acids (amino acids 269 and/or 265) are substituted binds to PLK1 competitively with wild-type IGFBP5, and thus, phosphorylation of wild-type IGFBP5 can be reduced in a mutant concentration-dependent manner.

The IGFBP5 mutant of the present invention may have S269 and T265 substituted with amino acids that cannot be phosphorylated by PLK1, specifically glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine or tryptophan, and the inventors of the present invention confirmed the effect using a mutant substituted with alanine.

The IGFBP5 mutant of the present invention may be contained in a vector that enables efficient introduction into a cell. The vector is preferably a vector, and both a viral vector and a non-viral vector can be used. Viral delivery mechanisms include, but are not limited to, lentivirus, retrovirus, adenovirus, herpes virus, and avipox virus. Non-viral delivery mechanisms may include lipid-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic surface amphiphiles, and combinations thereof. The lentivirus used as a viral vector for delivery of IGFBP5 shRNA and mutants in the present invention is a type of retrovirus and has the characteristic of being able to infect not only dividing cells but also undivided cells due to the nucleophilicity of a pre-integration complex (virus "shell") that enables active introduction into a nucleopore or a complete nuclear membrane.

Accordingly, the present invention provides a recombinant vector including a gene encoding the IGFBP5 mutation, and the vector is provided as a viral vector and can be used as an anticancer agent based on gene therapy.

In the present invention, a mutant means one or more amino acids constituting the IGFBP5 protein are substituted, and unless otherwise stated, a mutant having an anticancer effect by reducing the mobility and invasiveness of cancer cells and promoting apoptosis of cancer cells in the present specification means one in which the S269 and/or T265 amino acids of IGFBP5 are substituted. The term 'anticancer' used herein means an action of inhibiting the proliferation of cancer cells or killing them and an action of inhibiting or blocking the metastasis of cancer cells, and may refer to both prevention and treatment of cancer and may be used interchangeably therewith. The term 'prevention' used herein means any act of inhibiting the formation of cancer or delaying the onset of cancer by administering a composition, and 'treatment' means any act of improving or beneficially changing the symptoms of the disease by administering a composition.

In addition to the IGFBP5 expression inhibitor and IGFBP5 mutant described above, the anticancer agent of the present invention may additionally include other substances known to inhibit phosphorylation of IGFBP5 or reduce its expression, such as compounds, natural products, novel proteins, etc.

The anticancer agent of the present invention can be used, in particular, for the prevention and treatment of various solid cancers, such as colon cancer, cervical cancer, endometrial cancer, bladder cancer, esophageal cancer, kidney cancer, stomach cancer, ovarian cancer, breast cancer, and pancreatic cancer, in which the expression of IGFBP5 is excessive, and can also be used for the prevention and treatment of metastatic solid cancers caused by metastasis from primary cancer, and can be used for the prevention of metastatic cancer.

The pharmaceutical composition of the present invention may contain commonly used excipients, disintegrants, sweeteners, lubricants or flavoring agents, and may be formulated into tablets, capsules, powders, granules, suspensions, emulsions, syrups and other liquids by conventional methods. Specifically, the cell death activator of the present invention may be formulated into, for example, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs for oral administration. At this time, in order to be formulated into tablets and capsules, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin; an excipient such as dicalcium phosphate; a disintegrant such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax may be added. In addition, when formulated as a capsule, it may be manufactured by including one or more pharmaceutically acceptable carriers in addition to the substances mentioned above. For example, pharmaceutically acceptable carriers include saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and a mixture of one or more of these components, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition as an injectable formulation such as an aqueous solution, suspension, and emulsion, and an antibody or other ligand specific for a target cell may be combined with the above carrier so as to act specifically on the target cell. In addition, the composition can be preferably formulated for each disease or ingredient using an appropriate method in the relevant technical field or a method published in Remington's literature (Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA).

The composition of the present invention can be administered parenterally, and when administered parenterally, it is preferably administered by intravenous injection, intramuscular injection, or intrathoracic injection. In order to formulate for parenteral administration, the apoptosis-promoting anticancer agent can be mixed with a stabilizer or buffer in water to prepare a solution or suspension, and this can be formulated into a unit dosage form of an ampoule or vial.

It is preferable to select the dosage according to the absorption rate, inactivation rate and excretion rate of the active ingredient in the body, the age, sex and condition of the patient, and the severity of the disease to be treated. The anticancer agent promoting cancer cell death of the present invention may be administered once or several times daily in an amount of 1 to 100 mg, preferably 1 to 10 mg per 1 kg of body weight of an adult.

In the present invention, the amino acid sequence is described using the following abbreviations according to the IUPAC-IUB nomenclature.

Arginine (Arg, R), lysine (Lys, K), histidine (His, H), serine (Ser, S), threonine (Thr, T), glutamine (Gln, Q), asparagine (Asp, N), methionine (Met, M), leucine (Leu, L), isoleucine (Ile, I), valine (Val, V), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), alanine (Ala, A), glycine (Gly, G), proline (Pro, P), cysteine (Cys, C), aspartic acid (Asp, D) glutamic acid (Glu, E), norleucine (Nle)

The present invention can be modified in various ways and has various embodiments, and thus specific embodiments are illustrated in the drawings and described in detail in the detailed description below. However, this is not intended to limit the present invention to specific embodiments, and it should be understood that all modifications, equivalents, and substitutes included in the spirit and technical scope of the present invention are included. In describing the present invention, if it is determined that a specific description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are only intended to illustrate the present invention, and the content of the present invention is not limited by the following examples.

[Example]

<Example 1> Confirmation of the correlation between PLK1 and IGFBP5 expression levels and survival rate in adenocarcinoma through clinical data

In a clinical analysis targeting patients with adenocarcinoma, survival rates were analyzed according to PLK1 and IGFBP5 expression (Fig. 1a). The higher the stage of adenocarcinoma, the greater the difference in survival rates between patients with high and low expression of PLK1 and IGFBP5. The number of patients with increased IGFBP5 expression in tumor tissue compared to normal tissue was analyzed through heat map analysis (Fig. 1b). It was confirmed that the expression of IGFBP5 and PLK1 was high in the patient group with high stage. From the above results, it can be seen that both factors are correlated with metastasis.

<Example 2> Confirmation of IGFBP5 expression in NSCLC cells and confirmation of IGFBP5 expression level under EMT conditions induced by TGF-β treatment

We observed an increase in IGFPB5 expression in lung cancer cell lines A549 and H358 that were treated with TGF-β to induce metastasis through immunoblotting (Fig. 2a-Fig. 2b). We confirmed that the expression of IGFBP5 increased when treated with TGF-β. We performed real-time PCR to confirm the mRNA levels of IGFBP5 and EMT markers in lung cancer cell lines A549 and H358 that were treated with TGF-β to induce metastasis (Fig. 2c). The mRNA changes showed a similar pattern to the protein changes. From the above results, it can be seen that the expression level of IGFBP5 increases during the epithelial-mesenchymal transition induced by TGF-β treatment.

Next, the expression of IGFBP5, PLK1, and p-PLK1 was observed in non-small cell lung cancer cells by immunoblotting. The expression was observed in normal fibroblasts MRC5, primary lung cancer cells A549, and metastatic lung cancer cells H460, H358, and H1299, and it was confirmed that the expression of IGFBP5, PLK1, and p-PLK1 was higher in non-small cell lung cancer cells compared to MRC5 (Fig. 2d). The expression of IGFBP5, PLK1, and p-PLK1 was observed in normal fibroblasts, primary lung adenocarcinoma cells A549, stage 2 lung adenocarcinoma cells H522, stage 3A lung adenocarcinoma cells H1373, and stage 4 lung adenocarcinoma cells H2009. As a result of observation through immunoblot, IGFBP5 expression was the highest in A549 and H1373, and it was confirmed that when the expression of IGFBP5 was high, the expression of PLK1 and p-PLK1 was also high (Fig. 2e). After treating A549 and H358 with TGF-β to induce EMT, the expression of IGFBP5 was observed. In order to confirm whether EMT was well induced, the expression of EMT markers was also confirmed. After confirming that EMT was well induced, IGFBP5 expression was confirmed and it was confirmed that expression increased when EMT occurred (Fig. 2f). The immunoblot data were expressed numerically and presented as a graph (Fig. 2g).

<Example 3> Confirmation of the correlation between the expression level of IGFBP5 and survival rate in patients with solid tumors other than lung cancer

In order to confirm the clinical relevance of IGFBP5 in patients with solid cancers other than lung cancer, the overall survival rate of patients according to the expression level of IGFBP5 was confirmed through big data analysis (Fig. 3). It was confirmed that the survival rate of patients with high IGFBP5 expression in cervical cancer, endometrial cancer, and breast cancer was significantly lower than that of patients with low IGFBP5 expression (Fig. 3, A). In addition, when the survival rate of patients with bladder cancer, esophageal cancer, and ovarian cancer was analyzed, it was confirmed that the survival rate of patients was lower when IGFBP5 expression was high (Fig. 3, B). When the survival rate of patients with renal cancer and stomach cancer was analyzed, it was confirmed that it showed a similar trend to other solid cancers (Fig. 3, C).

<Example 4> Confirmation of phosphorylation of IGFBP5 by activated PLK1 in metastatic lung cancer cells induced by TGF-β

To investigate the interaction between PLK1 and IGFBP5 under the conditions of epithelial-mesenchymal transition induced by TGF-β treatment, immunoprecipitation was performed. PLK1 protein was precipitated with agarose beads and PLK1 antibody, and analyzed by immunoblotting. In A549 cells, IGFBP5 bound to PLK1 only in the experimental group in which cancer metastasis was induced by TGF-β. In H460 cells, IGFBP5 bound to PLK1 in the control group as well, and the binding of PLK1 and IGFBP5 increased in the experimental group in which cancer metastasis was induced by TGF-β (Fig. 4, A-B). The above results suggest that the interaction between PLK1 and IGFBP5 is more active in cancer cells during the metastatic process.

Next, kinase assay was performed to analyze the interaction method between the two proteins. To perform kinase assay, IGFBP5 was cloned into pGEX-4T-1 vector to obtain GST-tagged IGFBP5 (Fig. 4C). GST-IGFBP5 was purified through GST purification (Fig. 4D). To verify that PLK1, a serine/threonine kinase, phosphorylates IGFBP5 as a substrate, purified IGFBP5 wild and active PLK1-T210D were reacted with radiolabeled γ- ³² P-ATP and analyzed. IGFBP5 was phosphorylated more strongly by active PLK1-TD than by TCTP, a positive control (Fig. 4E).

<Example 5> Identification of residues of IGFBP5 phosphorylated by PLK1

Liquid chromatography mass spectrometry was performed to identify the residues of IGFBP5 phosphorylated by PLK1. Using this analysis method, Ser-179, Thr-265, and Ser-269 of IGFBP5 were predicted to be phosphorylated by PLK1 (Fig. 5a). By analyzing the three predicted phosphorylation sites of IGFBP5 and the IGFBP5 protein sequence, three sites similar to the PLK1 phosphorylation motif were substituted with alanine using the site-specific mutagenesis method to produce non-phosphorylated mutants (Fig. 5b).

The above-mentioned mutants were produced as GST-labeled proteins, purified, and subjected to kinase assay. Among the three predicted phosphorylation sites of IGFBP5, the phosphorylation levels of alanine mutations at Thr-265 and Ser-269 were significantly reduced compared to the original (Fig. 5c). From the results above, it was found that PLK1 interacts through the phosphorylation reaction of IGFBP5 Thr265 and Ser269. As a result of confirming Thr265 and Ser269 of IGFBP5 in various species, it was confirmed that Thr265 and Ser269 of IGFBP5 are conserved in higher animals including humans (Fig. 5d).

<Example 6> Establishment of a lentiviral system for the expression of phosphorylated and nonphosphorylated point mutants of IGFBP5. Evaluation of the epithelial-mesenchymal transition effect after selection of cells expressing phosphorylated and nonphosphorylated point mutant genes of IGFBP5 using lentivirus

To construct a lentivirus system for phosphorylated and non-phosphorylated gene expression of IGFBP5, the inventors of the present invention amplified the IGFBP5 circular (WT) plasmid by PCR using primers 5'- ATAAGAATGCGGCCGCATGGTGTTGCTCACC -3' (forward primer, SEQ ID NO: 9) and 5'- CGGAATTCTCACTCAACGTTGCTGCTGTC -3' (reverse primer, SEQ ID NO: 10) after cutting it with Not1 and EcoR1, and subcloned it into the vector pLVX-TRE3G-eRFP (Fig. 6A). To construct a lentivirus system, pCMV-VSV.G and pCMV-Δ8.2, pLVX-TRE3G-eRFP-Target or pLVX-Tet3G DNA were transfected into HEK293 cells to express the lentivirus, and the viruses were harvested and used in cancer cells. After transfection, the virus culture was harvested at 12-hour intervals for up to 72 hours, filtered through a 0.2 μm filter, and centrifuged at 17,000 rpm, 4°C, for 90 minutes. The supernatant was discarded, and the viruses harvested in TNE Buffer were harvested, stored at 4°C, and used for cancer cell infection from the next day.

In A549, RFP-IGFBP5 protein was expressed via the Tet-On 3G inducible system using lentivirus (Fig. 6B). To examine the effect of IGFBP5 protein containing point mutations on regulating epithelial-mesenchymal transition in cancer cells, cancer cells were cultured as follows to infect lung cancer cells with lentiviruses expressing phosphorylated and non-phosphorylated point mutants of IGFBP5.

To establish a stabilized cell line expressing the intact and phosphorylated and non-phosphorylated point mutants of IGFBP5 in lung cancer cell line A549, we first infected the pLVX-Tet3G expressing lentivirus and selected the infected cells by treating them with G418 for 5 days. The selected A549Tet3G cells were infected with lentiviruses expressing the intact and phosphorylated point mutants (T265D, S269D) and non-phosphorylated point mutants (T265A, S269A) of IGFBP5, and then treated with puromycin for 48 hours to establish a stabilized cell line. Specifically, 5 x 10 ⁴ lung cancer cells A549 were seeded, and 2 ug pCMV-VSV.G, 4 ug pCMV-Δ8.2, and 4 ug pLVX-TRE3G-eRFP-IGFBP5 were transfected the next day to establish IGFBP5-overexpressing cells. The established cells were treated with 2 ug/ml doxycycline to induce expression of the original and phosphorylated and non-phosphorylated point mutants of IGFBP5, and the mRNA and protein expression levels of each IGFBP5 point mutant were confirmed to be well expressed. For comparison, the EMT marker changes were observed for the control group (Mock; lentivirus-treated group in which the target gene is not expressed), the A549 lung cancer cell line administration group expressing IGFBP5 protein (WT), the A549 lung cancer cell line administration group expressing phosphorylated point mutant IGFBP5 protein (T265D, S269D), and the A549 lung cancer cell line administration group expressing non-phosphorylated point mutant IGFBP5 protein (T265A, S269A). As shown in Fig. 6 (CD) and Fig. 7 (A), it was confirmed through the mRNA expression levels and protein expression levels that the original and each point mutant form of IGFBP5 were well expressed. The expression of EMT markers was confirmed in lung cancer cells expressing phosphorylated point mutant IGFBP5 and cells expressing non-phosphorylated point mutant IGFBP5 through immunoblotting. In lung cancer cells expressing IGFBP5 of the Thr265 phosphorylation point mutant of IGFBP5, the expression of E-cadherin was decreased and the expression of Vimentin was increased compared to cells expressing the non-phosphorylated point mutant IGFBP5 (Fig. 6C). In lung cancer cells expressing IGFBP5 of the Ser269 phosphorylation point mutant of IGFBP5, the expression of Vimentin and N-cadherin was observed to be higher than cells expressing the non-phosphorylated point mutant IGFBP5 (Fig. 6D).

These results suggest that phosphorylation of IGFBP5 promotes epithelial-mesenchymal transition in lung cancer cells and is inhibited by a non-phosphorylated point mutant.

<Example 7> Comparison of metastatic and invasive potential of cancer cells expressing phosphorylated or nonphosphorylated point mutants of IGFBP5

Expression of EMT markers was confirmed in lung cancer cells expressing phosphorylated point mutants of IGFBP5 and non-phosphorylated point mutants of IGFBP5 using real-time PCR. Expression of RFP and IGFBP5 was confirmed to be similar in all mutants by RFP-IGFBP5 expression (Fig. 7A). Expression of CDH2, VIM, and SNAI1 was increased and expression of CDH1 was decreased in cells expressing IGFBP5 phosphorylated point mutants compared to cells expressing non-phosphorylated point mutants of IGFBP5.

To observe the effect of these point mutants on cell migration, a migration assay was performed in lung cancer cells A549 expressing intact, phosphorylated, or nonphosphorylated point mutants of IGFBP5.

Specifically, 5 × 10 ⁴ lung cancer cells A549 expressing each of the original, phosphorylated, or non-phosphorylated point mutant proteins of IGFBP5 were seeded in an 8.0 μm, 24-well insert, and the inset was placed in a 24-well plate containing medium containing 10% fetal bovine serum (FBS). For the positive control, 0.5 ml of RPMI 1640 (10% FBS) containing 5 ng/ml TGF-β was used. 48 hours after cell seeding, 500 μl of 4% paraformaldehyde was dispensed, washed three times with 1X PBS, and stained with 0.05% crystal-violet solution for 5 minutes. After 5 minutes, the cells were washed five times with 1X PBS, and the staining intensity was measured using an Odyssey infrared imaging system. The relative staining intensity of each experimental group was calculated and displayed on a graph, when the staining intensity of the control group was set to 1.

As a result of the study, in the experimental group expressing the IGFBP5 phosphorylation point mutant protein, the staining intensity value increased up to 5.5 times compared to the control group, which was similar to or higher than the TGF-β treatment group, which was the positive control group, and it was observed that the staining intensity was relatively lower in the non-phosphorylation point mutant experimental group than in the IGFBP5 phosphorylation point mutant (Fig. 7B). These results indicate that the IGFBP5 phosphorylation point mutant promotes the mobility of lung cancer cells, whereas the IGFBP5 non-phosphorylation point mutant exhibits the effect of inhibiting the mobility of lung cancer cells.

To observe the effect of these point mutants on the invasiveness of cancer cells, an invasion assay using matrigel was performed in lung cancer cells A549 expressing intact, phosphorylated, or nonphosphorylated point mutants of IGFBP5.

Specifically, after completely melting the matrigel at 4℃ for 16-20 hours, the matrigel was diluted to 1 mg/ml with cold serum-free RPMI 1640 (4℃). 100 μl of the matrigel mixture (1 mg/ml) was added to an 8.0 μm 24-well insert and solidified in a 37℃ incubator for 12-20 hours. 2 × 10 cells/well of lung cancer cells A549 expressing each IGFBP5 native, phosphorylated, or non-phosphorylated point mutant protein were diluted in serum-free RPMI 1640 (36℃) and dispensed into each insert. 0.5 ml/well of warm RPMI 1640 (10% FBS) at 36℃ was dispensed here. For the positive control group, 0.5 ml of 36℃ RPMI 1640 (10% FBS) containing 5 ng/ml TGF-β was aliquoted and used. The medium was changed once every 3 days and the degree of invasion was observed. On the 5th day, when sufficient cancer cell invasion was observed, the medium was removed, washed with 1X PBS, and the cells inside the insert were scraped with a cotton swab and washed with 1X PBS to remove any remaining cells and matrigel inside the insert. 500 ul of 4% paraformaldehyde was aliquoted into 24 wells with the outer surface of the insert, incubated at room temperature for 5 minutes, washed three times with 1X PBS, and stained with 0.05% crystal-violet solution for 5 minutes. After 5 minutes, it was washed five times with 1X PBS, and the degree of staining was dissolved in DMSO and measured for a wavelength at 590 nm. When the absorbance of the control group is set to 1, the relative absorbance of each experimental group was calculated and displayed on a graph.

As a result of the study, in the experimental group expressing the IGFBP5 phosphorylation point mutant protein, the increase was 5.7- to 8-fold compared to the control group, and it was observed that the relative absorbance of the IGFBP5 non-phosphorylation point mutant experimental group was lower than that of the IGFBP5 phosphorylation point mutant (Fig. 7C). Therefore, it was found that the IGFBP5 non-phosphorylation point mutant exhibited an effect of inhibiting the mobility and invasiveness of lung cancer cells.

Cell viability was observed in cells expressing IGFBP5 phosphorylated or nonphosphorylated point mutants using a caspase-3 assay. Caspase-3 activity was significantly increased and cell viability was decreased in the nonphosphorylated point mutants of Thr265 and Ser269 (Fig. 7D). From the results, it was confirmed that when Thr265 and Ser269 of IGFBP5 were phosphorylated, cell viability was increased, whereas the nonphosphorylated point mutant of IGFBP5 induced apoptosis through caspase-3 activity.

<Example 8> Selection of IGFBP5 shRNA

In order to confirm the effect of inhibiting the mRNA expression of IGFBP5, shRNA and lentivirus containing it were produced. Specifically, to inhibit the mRNA expression of IGFBP5, primers were produced to produce shRNA targeting the nucleotide sequence at positions 443-463 or 467-487 or 674-694 of the human IGFBP5 mRNA (Human IGFBP5 mRNA [NM_ 000599.4], SEQ ID NO: 5). The sense region of the 443-463 nucleotide target sequence is The pLKO-puro.1-IGFBP5 plasmid was constructed using the pLKO-puro.1 vector based on the 5'- AGG CTG AAG CAG TGA AGA AGG -3' (SEQ ID NO: 11) as the sense region and 5'- CCT TCT TCA CTG CTT CAG CCT -3' (SEQ ID NO: 12) as the antisense region. 5'- CCGG - AGGCTGAAGCAGTGAAGAAGG - CTCGAG - CCTTCTTCACTGCTTCAGCCT - TTTTTG -3' (SEQ ID NO: 13) was used as the forward primer, and 5'- AATTCAAAAA - AGGCTGAAGCAGTGAAGAAGG - CTCGAG - CCTTCTTCACTGCTTCAGCCT -3' (SEQ ID NO: 14) was used as the reverse primer. The sense region of the shRNA targeting the 467-487 nucleotide sequence is The pLKO-puro.1-IGFBP5 plasmid was constructed using the pLKO-puro.1 vector based on the 5'- GCA GAA AGA AGC TGA CCC AGT -3' (SEQ ID NO: 15) as the sense region and 5'- ACT GGG TCA GCT TCT TTC TGC -3' (SEQ ID NO: 16) as the antisense region. 5'-CCGG - GCAGAAAGAAGCTGACCCAGT-CTCGAG- ACTGGGTCAGCTTCTTTCTGC-TTTTTG -3' (SEQ ID NO: 17) was used as the forward primer, and 5'-AATTCAAAAA-GCAGAAAGAAGCTGACCCAGT-CTCGAG- ACTGGGTCAGCTTCTTTCTGC -3' (SEQ ID NO: 18) was used as the reverse primer. The sense region of the shRNA targeting the 674-694 nucleotide sequence is The pLKO-puro.1-IGFBP5 plasmid was constructed using the pLKO-puro.1 vector based on the 5'- ACA AGA GAA AGC AGT GCA AAC -3' (SEQ ID NO: 19) as the sense region and 5'- GTT TGC ACT GCT TTC TCT TGT -3' (SEQ ID NO: 20) as the antisense region. 5'-CCGG - ACAAGAGAAAGCAGTGCAAAC-CTCGAG- GTTTTGCACTGCTTTCTCTTGT-TTTTTG -3' (SEQ ID NO: 21) was used as the forward primer, and 5'-AATTCAAAAA-ACAAGAGAAAGCAGTGCAAAC-CTCGAG- GTTTGCACTGCTTTCTCTTGT -3' (SEQ ID NO: 22) was used as the reverse primer. The loop sequence is 5'-CTCGAG-3' (SEQ ID NO: 23), and shRNA was produced in the form included in the primer. This was expressed through HEK293 cell transfection with pHR'-CMV-VSVG and pHR'-CMV-deltaR8.2, and the cell culture medium was collected to produce lentivirus. The lentivirus was concentrated using a centrifuge. To confirm virus expression, A549 cells were cultured at ^5X104 /ml, and the next day, lentivirus was added to 20 ml/well in infection buffer (Infection Buffer; 10 mM HEPES (Sigma-Aldrich, USA), 1 mg/ml polybrene (Sigma-Aldrich, MO, USA)) to infect cancer cells. After 24 hours, the infected cells were treated with puromycine (Sigma-Aldrich, MO, USA) for 48 hours to select live, infected cells that did not die. RNA from the selected cells was collected and real-time polymerase chain reaction (real-time PCR) was used to observe that the expression of IGFBP5 was suppressed.

As shown in Fig. 8A, the expression of IGFBP5 mRNA in lung cancer cells A549 infected with shIGFBP5 decreased compared to cells infected with the control shRNA (shCtrl), indicating that the expression of IGFBP5 was inhibited. Subsequent experiments were conducted using shRNA targeting the #443 site, which had the greatest inhibitory effect among the three produced shRNAs.

<Example 9> Confirmation of inhibition of epithelial-mesenchymal transition progression by IGFBP5 shRNA

Next, to evaluate the reduction in epithelial-mesenchymal transition due to inhibition of IGFBP5 expression, the inventors inhibited the expression of IGFBP5 using IGFBP5 shRNA in metastatic cells treated with TGF-β and observed epithelial-mesenchymal transition markers at the protein level.

Specifically, lung cancer cells A549 were cultured at ^5X104 cells/ml and infected with control virus (shCtrl) or shIGFBP5, then treated with puromycine (Sigma-Aldrich, MO, USA) for 48 hours to select viable cells, and then seeded at ^5X104 cells/ml again, and the cells were collected the next day after treatment with 5 ng/ml TGF-β for 48 hours. The harvested cells were treated with 100 ㎕ of a pulverization solution (0.5% Triton X-100, 20 mM Tris, pH 7.5, 2 mM MgCl ₂ , 1 mM DTT, 1 mM EGTA, 50 mM beta-glycerophosphoate, 25 mM NaF, 1 mM Na ₃ VO ₄ , 2 ㎍/ml leupeptin, 2 ㎍/ml pepstatin A, 100 ㎍/ml PMSF, 1 ㎍/ml antipain), and then protein quantification was performed. The quantified proteins were electrophoresed through SDS-PAGE, and immunoblotting was performed using an epithelial-mesenchymal transition marker antibody to confirm the decrease in the epithelial-mesenchymal transition process (BC in Fig. 8). In addition, mRNA was purified from the collected cells, cDNA was synthesized, and real-time polymerase chain reaction (real-time PCR) was performed. By observing that epithelial-mesenchymal transition markers such as N-cadherin, Vimentin, and SNAI1 at the mRNA level decreased, and E-Cadherin increased, it was confirmed that IGFBP5 shRNA reduces the epithelial-mesenchymal transition process in a TGF-β-induced metastatic environment (Fig. 8D).

<Example 10> Evaluation of the inhibitory effect on mobility and invasiveness by IGFBP5 shRNA and the increased effect on mobility and invasiveness by phosphorylation point mutants in a metastatic environment

In addition, the present inventors demonstrated the effect of IGFBP5 shRNA on suppressing cell mobility using an insert in a metastatic environment.

)에 희석하여 인서트에 분주하였다. 인서트 밖의 24 well에는 RPM1(10% FBS 포함)을 0.5 ml/well씩 분주하고 48시간 후 4% 파라포름알데히드(paraformaldehyde) 500 ㎕를 분주하고 1XPBS로 3회 세척하여 0.05% crystal-violet 용액으로 5분간 염색하였다. 5분 후 1XPBS로 5회 세척하였고, 염색된 정도를 590nm에서 파장을 측정하였다. 대조군의 흡광도를 1이라고 할 때 각 실험군에서의 상대적 흡광도를 산출하여 그래프를 표시하였다(도 9에 A).Specifically, lung cancer cells A549 were infected with control shRNA (shCtrl) or IGFBP5 shRNA (shIGFBP5) viruses. The infected cells were seeded in 24-well inserts at a cell number of ^5X104 cells/well with serum-free RPM1 (36

) and dispensed into the insert. RPM1 (containing 10% FBS) was dispensed at 0.5 ml/well into the 24 wells outside the insert, and after 48 hours, 500 ㎕ of 4% paraformaldehyde was dispensed, washed three times with 1X PBS, and stained with 0.05% crystal-violet solution for 5 minutes. After 5 minutes, the cells were washed five times with 1X PBS, and the degree of staining was measured at a wavelength of 590 nm. When the absorbance of the control group was 1, the relative absorbance in each experimental group was calculated and displayed on a graph (Fig. 9A).

As a result, as shown in A in Fig. 9, when the expression of IGFBP5 was suppressed by infection with shIGFBP5, cell mobility was significantly reduced compared to cells infected with the control virus (shCtrl). A similar trend was observed when mobility was observed after TGF-β treatment (A in Fig. 9).

Next, the present inventors demonstrated the effect of IGFBP5 shRNA in suppressing cell invasiveness using Matrigel in a metastatic environment.

배양기에서 12-20시간 동안 굳혀주었다. 굳은 마트리겔 인서트(Matrigel insert)에 각각 shRNA(shCtrl) 또는 IGFBP5 shRNA(shIGFBP5) 바이러스를 감염시킨 세포를 2X10 ⁵ cells/well의 세포수로 혈청프리 RPM1(36

)에 희석하여 인서트에 분주하였다. 여기에 36

의 따뜻한 RPM1(10% FBS 포함)을 0.5 ml/well씩 분주하였다. 이 후 3일에 한 번씩 배지를 교환해주고 침습되는 정도를 관찰하였으며, 암세포의 침습이 충분히 일어난 것으로 관찰된 7일 차에 배지를 제거하고 1XPBS로 세척한 후, 인서트 안쪽의 세포들을 면봉으로 긁어 내었고, 1XPBS로 세척하여 인서트 내부에 세포와 마트리겔(Matrigel)의 잔여물이 남지 않도록 제거하였다. 인서트 바깥 면이 있는 24 well에 4% 파라포름알데히드(paraformaldehyde) 500 ㎕를 분주하고 5분간 실온에서 인큐베이션한 후 5분에 1XPBS로 3회 세척하여, 0.05% crystal-violet 용액으로 5분간 염색하였다. 5분 후 1XPBS로 5회 세척하였고, 염색된 정도를 590nm에서 파장을 측정하였다. 대조군의 흡광도를 1이라고 할 때 각 실험군에서의 상대적 흡광도를 산출하여 그래프를 표시하였다 (도 9에 B).For the present invention, Matrigel was completely dissolved at 4°C for 16 to 20 hours, and then Matrigel was diluted with cold serum-free MEM (4°C) to 1 mg/ml. 1 ml of Matrigel mixture (1 mg/ml) was placed in an 8.0 mm 24-well insert and incubated at 37°C.

The cells were solidified in an incubator for 12-20 hours. The cells infected with shRNA (shCtrl) or IGFBP5 shRNA (shIGFBP5) virus were seeded in serum-free RPM1 (36) at a cell number of ^2X105 cells/well on the solidified Matrigel insert.

) was diluted and dispensed into the insert. Here, 36

Warm RPM1 (containing 10% FBS) was dispensed at 0.5 ml/well. The medium was changed once every 3 days, and the degree of invasion was observed. On the 7th day, when sufficient cancer cell invasion was observed, the medium was removed, washed with 1X PBS, and the cells inside the insert were scraped with a cotton swab and washed with 1X PBS to remove any remaining cells and Matrigel inside the insert. 500 ㎕ of 4% paraformaldehyde was dispensed into 24 wells with the outer surface of the insert, incubated at room temperature for 5 minutes, washed three times with 1X PBS every 5 minutes, and stained with 0.05% crystal-violet solution for 5 minutes. After 5 minutes, the medium was washed five times with 1X PBS, and the degree of staining was measured at a wavelength of 590 nm. When the absorbance of the control group is set to 1, the relative absorbance of each experimental group was calculated and displayed on a graph (B in Figure 9).

As a result, as shown in B in Fig. 9, when the expression of IGFBP5 was suppressed by infection with shIGFBP5, the invasiveness of the cells was significantly reduced compared to cells infected with the control virus (shCtrl) (B in Fig. 9).

Lung cancer cells A549 were cultured at ^5X104 cells/ml and infected with control virus (shCtrl) or shIGFBP5, then treated with puromycine (Sigma-Aldrich, MO, USA) for 48 hours to select viable cells, and then seeded at ^5X104 cells/ml again, and the cells were collected the next day after treatment with 5 ng/ml TGF-β for 48 hours. The harvested cells were treated with 100 ㎕ of pulverization solution (0.5% Triton X-100, 20 mM Tris, pH 7.5, 2 mM MgCl ₂ , 1 mM DTT, 1 mM EGTA, 50 mM beta-glycerophosphoate, 25 mM NaF, 1 mM Na ₃ VO ₄ , 2 ㎍/ml leupeptin, 2 ㎍/ml pepstatin A, 100 ㎍/ml PMSF, 1 ㎍/ml antipain), and then protein quantification was performed. Caspase-3 activity was observed in the quantified protein through a caspase-3 assay, and it was confirmed that caspase-3 activity was significantly increased in cells infected with shIGFBP5 (Fig. 9C).

In addition to when IGFBP5 was inhibited, it was demonstrated that when IGFBP5 was phosphorylated, it increased the mobility and invasiveness of lung cancer cells. As a result of evaluating the mobility and invasiveness of MOCK, IGFBP5 WT, phosphorylation point mutants, and non-phosphorylation point mutants using inserts and Matrigel under the above conditions, it was confirmed that cell mobility and invasiveness increased in the phosphorylation point mutants compared to WT and non-phosphorylation point mutants (Fig. 9D-E).

When the results of Fig. 9 are summarized, it was demonstrated that when the expression of IGFBP5 was suppressed in lung cancer cells using IGFBP5 shRNA in a metastatic environment, the epithelial-mesenchymal transition was reduced, and cell motility and invasiveness were also reduced. It was confirmed that cell viability decreased as the expression of IGFBP5 decreased, proving that IGFBP5 affects cell survival. In addition, it was demonstrated that when IGFBP5 was phosphorylated, it increased cell motility and invasiveness.

<Example 11> Evaluation of the effect of IGFBP5 shRNA on cell viability reduction in solid cancer

Cervical cancer HeLa cells were cultured at ^2X104 cells/ml and breast cancer BT-20 cells were cultured at ^4X104 cells/ml and infected with control virus (shCtrl) or shIGFBP5, then treated with puromycine (Sigma-Aldrich, MO, USA) for 48 hours to select living cells. Then, HeLa cells were divided again at ^2X104 cells/ml and BT-20 cells were divided again at ^4X104 cells/ml and the cells were collected the next day after treating with 5 ng/ml TGF-β for 48 hours. The harvested cells were treated with 100 ㎕ of pulverization solution (0.5% Triton X-100, 20 mM Tris, pH 7.5, 2 mM MgCl ₂ , 1 mM DTT, 1 mM EGTA, 50 mM beta-glycerophosphoate, 25 mM NaF, 1 mM Na ₃ VO ₄ , 2 ㎍/ml leupeptin, 2 ㎍/ml pepstatin A, 100 ㎍/ml PMSF, 1 ㎍/ml antipain), and then protein quantification was performed. As shown in Fig. 10A, the expression of IGFBP5 mRNA was decreased in cervical cancer cells HeLa and breast cancer cells BP-20 infected with shIGFBP5 compared to cells infected with the control shRNA (shCtrl), indicating that the expression of IGFBP5 was inhibited (Fig. 10A). Caspase-3 activity was observed through a caspase-3 assay with the quantified protein, and it was confirmed that caspase-3 activity significantly increased in HeLa cells and BT-20 cells infected with shIGFBP5 under conditions in which caspase-3 activity increased in cells treated with paclitaxel, which was used as a positive control (B in Fig. 10). This proved that IGFBP5 affects the survival of cervical cancer cells.

We also observed whether there was a change in survival rate by shIGFBP5 treatment in the metastatic environment induced by TGF-β treatment. As a result, the expression of IGFBP5 mRNA decreased in HeLa cells infected with shIGFBP5 compared to cells infected with the control shRNA (shCtrl), and the expression of IGFBP5 mRNA increased in cells infected with shRNA (shCtrl) and then treated with TGF-β (Fig. 10C). Caspase-3 activity was observed through a caspase-3 assay with the quantified protein, and it was confirmed that caspase-3 activity increased significantly in cells infected with shIGFBP5, and it was also confirmed that caspase-3 activity increased significantly in cells infected with shIGFBP5 when treated with TGF-β (Fig. 10D). This demonstrated that IGFBP5 affects the survival of cervical cancer cells even in the metastatic environment. Through the above, it was confirmed that the survival rate decreased as the expression of IGFBP5 decreased in lung cancer cells, cervical cancer cells, and breast cancer cells.

Although the embodiments have been described with limited drawings as described above, those skilled in the art can apply various technical modifications and variations based on the above. For example, even if the described techniques are performed in a different order than the described method, and/or the components of the described system, structure, device, circuit, etc. are combined or combined in a different form than the described method, or are replaced or substituted by other components or equivalents, appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents to the claims are also included in the scope of the claims described below.

Table 1 below shows the sequence information of proteins and oligonucleotides used in the description of the invention.

Sequence number 1 IGFBP5 wt mvlltavlll laayagpaqs lgsfvhcepc dekalsmcpp splgcelvke pgcgccmtca laegqscgvy tercaqglrc lprqdeekpl hallhgrgvc lneksyreqv kierdsrehe epttsemaee tyspkifrpk htriselkae avkkdrrkkl tqskfvggae ntahpriisa qg pcrrhmeasl qelkasprmv pravylpncd rkgfykrkqc kpsrgrkrgi cwcvdkygmk lpgmeyvdgd fqchtfdssn ve Sequence number 2 IGFBP5_265/269 MVLLTAVLLL LAAYAGPAQS LGSFVHCEPC DEKALSMCPP SPLGCELVKE PGCGCCMTCA LAEGQSCGVY TERCAQGLRC LPRQDEEKPL HALLHGRGVC LNEKSYREQV KIERDSREHE EPTTSEMAEE TYSPKIFRPK HTRISELKAE AVKKDRRKKL TQSKFVGGAE NTAHPRIISA PEMRQESEQG QELKASPRMV PRAVYLPNCD RKGFYKRKQC KPSRGRKRGI CWCVDKYGMK LPGMEYVDGD FQCHXFDSXN VE Sequence number 3 IGFBP5 Thr265 MVLLTAVLLL LAAYAGPAQS LGSFVHCEPC DEKALSMCPP SPLGCELVKE PGCGCCMTCA LAEGQSCGVY TERCAQGLRC LPRQDEEKPL HALLHGRGVC LNEKSYREQV KIERDSREHE EPTTSEMAEE TYSPKIFRPK HTRISELKAE AVKKDRRKKL TQSKFVGGAE NTAHPRIISA PEMRQESEQG QELKASPRMV PRAVYLPNCD RKGFYKRKQC KPSRGRKRGI CWCVDKYGMK LPGMEYVDGD FQCHXFDSSN VE Sequence number 4 IGFBP5 Ser269 MVLLTAVLLL LAAYAGPAQS LGSFVHCEPC DEKALSMCPP SPLGCELVKE PGCGCCMTCA LAEGQSCGVY TERCAQGLRC LPRQDEEKPL HALLHGRGVC LNEKSYREQV KIERDSREHE EPTTSEMAEE TYSPKIFRPK HTRISELKAE AVKKDRRKKL TQSKFVGGAE NTAHPRIISA PEMRQESEQG QELKASPRMV PRAVYLPNCD RKGFYKRKQC KPSRGRKRGI CWCVDKYGMK LPGMEYVDGD FQCHTFDSXN VE Sequence number 5 IGFBP5 gene atggtgttgctcaccgcggtcctcctgctgctggccgcctatgcggggccggcccagagcctgggctccttcgtgcactgcgagccctgcgacgagaaagccctctccatgtgcccccccagccccctgggctgcgagctggtcaaggagccgggctgcggctgctgcatgacctgcgccctggccgaggggca gtcgtgcggc gtctacaccgagcgctgcgcccaggggctgcgctgcctcccccggcaggacgaggagaagccgctgcacgccctgctgcacggccgcggggtttgcctcaacgaaaagagctaccgcgagcaagtcaagatcgagagagactcccgtgagcacgaggagcccaccacctctgagatggccgaggagacctactcccccaagatct tccggcccaaacacacccgcatctccgagctgaaggctgaagcagtgaagaaggaccgcagaaagaagctgacccagtccaagtttgtcgggggagccgagaacactgcccaccccccggatcatctctgcacctgagatgagacaggagtctgagcagggcccctgccgcagacacatggaggcttccctgcaggagctcaaagccagcccacg catggtgccccgtgctgtgtacctgcccaattgtgaccgcaaaggattctacaagagaaagcagtgcaaaccttcccgtggccgcaaacgtggcatctgctggtgcgtggacaagtacgggatgaagctgccaggcatggagtacgttgacggggactttcagtgccacaccttcgacagcagcaacgttgagtga Sequence number 6 IGFBP5 shRNA #443 AGG CTG AAG CAG TGA AGA AGG CTCGAG CCT TCT TCA CTG CTT CAG CCT Sequence number 7 IGFBP5 shRNA #467 GCA GAA AGA AGC TGA CCC AGT CTCGAG ACT GGG TCA GCT TCT TTC TGC Sequence number 8 IGFBP5 shRNA #674 ACA AGA GAA AGC AGT GCA AAC CTCGAG GTT TGC ACT GCT TTC TCT TGT Sequence number 9 forward primer for amplifying IGFBP5 wildtype plasmid ATAAGAATGCGGCCGCATGGTGTTGCTCACC Sequence number 10 reverse primer for amplifying IGFBP5 wildtype plasmid CGGAATTCTCACTCAACGTTGCTGCTGTC Sequence number 11 Sense region_targeting 443-463 region AGG CTG AAG CAG TGA AGA AGG Sequence number 12 antisense region_targeting 443-463 region CCT TCT TCA CTG CTT CAG CCT Sequence number 13 forward primer for amplifying pLKO-puro.1 vetor which is encoding shRNA targeting 443-463 region of IGFBP5 CCGGAGGCTGAAGCAGTGAAGAAGGCTCGAGCCTTCTTCACTGCTTCAGCCTTTTTTG
Sequence number 14 reverse primer for amplifying pLKO-puro.1 vetor which is encoding shRNA targeting 443-463 region of IGFBP5 AATTCAAAAAAAGGCTGAAGCAGTGAAGAAGGCTCGAGCCTTCTTCACTGCTTCAGCCT Sequence number 15 Sense region_targeting 467-487 region GCA GAA AGA AGC TGA CCC AGT Sequence number 16 antisense region_targeting 467-487 region ACT GGG TCA GCT TCT TTC TGC Sequence number 17 forward primer for amplifying pLKO-puro.1 vetor which is encoding shRNA targeting 467-487 region of IGFBP5 CCGGGCAGAAAGAAGCTGACCCAGT-CTCGAG-ACTGGGTCAGCTTCTTTCTGC-TTTTTG Sequence number 18 reverse primer for amplifying pLKO-puro.1 vetor which is encoding shRNA targeting 467-487 region of IGFBP5 AATTCAAAAA-GCAGAAAGAAGCTGACCCAGT-CTCGAG- ACTGGGTCAGCTTCTTTCTGC Sequence number 19 Sense region_targeting 674-694 region ACA AGA GAA AGC AGT GCA AAC Sequence number 20 antisense region_targeting 674-694 region GTT TGC ACT GCT TTC TCT TGT Sequence number 21 forward primer for amplifying pLKO-puro.1 vetor which is encoding shRNA targeting 674-694 region of IGFBP5 CCGGACAAGAGAAAGCAGTGCAAAC-CTCGAG-GTTTGCACTGCTTTCTCTTGT-TTTTTG Sequence number 22 reverse primer for amplifying pLKO-puro.1 vetor which is encoding shRNA targeting 674-694 region of IGFBP5 AATTCAAAAA-ACAAGAGAAAGCAGTGCAAAC-CTCGAG- GTTTGCACTGCTTTCTCTTGT Sequence number 23 Loop sequence of the shRNAs CTCGAG

Attach electronic file of sequence list

Claims (11)

A pharmaceutical composition for preventing or treating cancer, comprising RNA for inhibiting IGFBP5 expression or a vector expressing said RNA. In the first paragraph,
The above RNA for suppressing IGFBP5 expression is one type selected from the group consisting of aptamer, siRNA, shRNA, and miRNA.
A pharmaceutical composition, wherein the RNA for inhibiting IGFBP5 expression comprises a base sequence complementary to the base sequence of SEQ ID NO: 11, SEQ ID NO: 15, or SEQ ID NO: 19. In the first paragraph,
The above RNA for suppressing IGFBP5 expression is shRNA.
A pharmaceutical composition, wherein the shRNA comprises a base sequence complementary to one base sequence selected from the group consisting of SEQ ID NO: 6 to SEQ ID NO: 8. In the first paragraph,
The above RNA for suppressing IGFBP5 expression is shRNA.
A pharmaceutical composition, wherein the vector expressing the shRNA comprises at least one base sequence selected from the group consisting of SEQ ID NO: 6 to SEQ ID NO: 8. In the first paragraph,
The above composition is a pharmaceutical composition that inhibits cancer metastasis by reducing the mobility and invasiveness of cancer cells. In the first paragraph,
A pharmaceutical composition, wherein the composition inhibits tumor formation ability. In the first paragraph,
A pharmaceutical composition, wherein the composition further comprises an IGFBP5 mutant protein comprising an amino acid sequence of sequence number 2. In the first paragraph,
A pharmaceutical composition for preventing or treating cancer, characterized in that the cancer is a solid cancer. In the first paragraph,
A pharmaceutical composition for preventing or treating cancer, characterized in that the solid cancer is at least one cancer selected from the group consisting of glioblastoma, ovarian cancer, bladder cancer, non-functioning pituitary adenoma, esophageal cancer, kidney cancer, breast cancer, stomach cancer, rectal cancer, cervical cancer, endometrial cancer, lung cancer, and pancreatic cancer. In the first paragraph,
A pharmaceutical composition for preventing or treating cancer, characterized in that the lung cancer is non-small cell lung cancer. In the first paragraph,
A pharmaceutical composition for preventing or treating cancer, characterized in that the non-small cell lung cancer is adenocarcinoma.