CD133 related to anticancer agent resistance in colon cancer and use thereof

Abstract

The present invention relates to a use of CD133 involved in resistance to an EGFR-targeting agent in colon cancer. The CD133 protein may be used as a novel target protein for diagnosing and treating resistant cancer as well as general cancer.

Claims

1. A method of treating resistance to Gefitinib in colon cancer, the method comprising administering an inhibitor against a CD133 gene to a subject resistant to Gefitinib, wherein the inhibitor comprises an antisense oligonucleotide, siRNA, or shRNA against the CD133 gene, or a vector comprising the inhibitor.

Description

DESCRIPTION OF DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) FIGS. 1A-1E show the result of identifying a CD133 expression regulatory mechanism in liver cancer cells: (A) the change in CD133 expression according to EGF expression; (B-C) the change in CD133 expression according to an EGF downstream signaling molecule, NF-KB; and (D-E) the relationship between NF-κB and CD133, confirmed by promoter activity.

(3) FIGS. 2A-2E show the result of confirming that CD133 is involved in promotion of microvesicle budding in liver cancer cells: (A) confirmation of the relationship between an EGF signaling system and microvesicle and TNT formation; (B) confirmation of microvesicle and TNT formation in a CD133-overexpressing cell line; and (C-E) confirmation of the change in activation of Rac1 and RhoA associated with microvesicle budding and a downstream signaling factor, Erk1/2, of RhoA according to CD133 expression

(4) FIGS. 3A-3B show the result of analyzing microvesicles budded from cancer cells: (A) the result of confirming whether CD133 is included in microvesicles budded from various cancer cells; and (B) the result of observing the migration of microvesicles containing CD133 to adjacent cells.

(5) FIGS. 4A-4F show the result of confirming that CD133 is involved in oncogenic protein transport and microvesicle budding in colon cancer: (A) confirmation whether an oncogenic protein is contained in microvesicles according to CD133 expression; (B-C) confirmation that CD133 expression contributes to microvesicle budding and a size change; (D) confirmation that CD133 expression contributes to the transport of an oncogenic protein via microvesicles and cell proliferation; and (E-F) confirmation that the transport of an oncogenic protein via microvesicles contributes to the change in cell migration to and invasiveness of adjacent normal cells.

(6) FIGS. 5A-5F show the result of confirming that CD133-containing microvesicles induce anticancer agent resistance (gefitinib) in colon cancer: (A-B) confirmation of the capability of CD133-containing microvesicles to induce anticancer agent resistance (gefitinib) by confirmation of the proliferation of cancer cells; (C-D) the result of confirming that cancer cell proliferation is caused by an oncogenic protein transported by microvesicles through the change in KRAS downstream signaling molecule and target gene expression; and (E-F) confirmation that cell migration to and invasiveness of adjacent normal cells are changed by the oncogenic protein transported by CD133-containing microvesicles in the presence of gefitinib.

MODES OF THE INVENTION

(7) Hereinafter, the advantages and features of the present invention and the methods of accomplishing the same may be clearly understood with reference to the detailed description of exemplary embodiments and the accompanying drawings. However, the present invention is not limited to the exemplary embodiments disclosed below, and may be embodied in many different forms. These exemplary embodiments are merely provided to complete the disclosure of the present invention and fully convey the scope of the present invention to those of ordinary skill in the art, and the present invention should be defined by only the accompanying claims.

EXAMPLE 1

Confirmation of CD133 Expression Regulatory Mechanism in Cancer Cells

(8) An epithelial growth factor (EGF) signaling system has been reported to be significant in the occurrence and development of cancer. CD133 (NCBI Gene ID: 8842) is known as a marker of a cancer stem cell, and particularly, to play a crucial role in formation of liver cancer stem cells. Therefore, the correlation between the EGF signaling system and CD133 expression in a liver cancer cell line was investigated.

(9) After treatment with an inhibitor against an EGF downstream signaling molecule, CD133 expression was investigated. Specifically, for transfection and Western blotting, a specific gene (plasmid vector or siRNA) was expressed in cells using a transfection reagent. For general plasmid vector transfection, 24 hours after transfection, cells were harvested, and for siRNA transfection, 48 hours after transfection, cells were harvested. For Western blotting, the harvested cells were lysed in RIPA buffer, and centrifuged at 12,000 g for 20 minutes at 4° C., followed by collecting a supernatant. The collected supernatant was subjected to SDS-PAGE gel electrophoresis, and transferred onto a nitrocellulose membrane, followed by detecting the expression of a desired protein using suitable antibodies. As a result, it was observed that NF-κB is involved in EGF-induced CD133 expression (FIG. 1B). It was confirmed that, when the expression of NF-κB subunits, such as p50 (NCBI Gene ID: 4790) and p65 (NCBI Gene ID: 5970), was inhibited, CD133 expression decreases (FIG. 1C). Promoter activity was observed in a promoter near CD133 ORF. Specifically, to measure the promoter activity, a CD133 ORF promoter region was cloned in a special vector expressing luciferase. Cells were simultaneously transfected with the luciferase vector and a Renilla plasmid vector for quantification. Twenty-four hours after transfection, the cells were treated with EGF for 14 hours, lysed with lysis buffer and centrifuged to obtain a supernatant, and then luciferase activity was measured using the Luminometer 20/20 (FIG. 1D). In a promoter which had been subjected to treatment with an NF-κB inhibitor and an NF-κB subunit-binding site, promoter activity was not observed (FIG. 1D). Taken together, it was confirmed that EGF activates NF-κB and regulates CD133 expression at the transcription level (FIG. 1A).

EXAMPLE 2

Identification of CD133 Role in Cancer Cells

(10) The relationship between EGF and microvesicle and TNT formation was investigated by microscopic analysis. Specifically, after 14 hours of EGF treatment, cells were fixed with 4% paraformaldeyde and treated with WGA-488 and DAPI for 10 minutes to stain the cell membrane and nucleus. Fluorescence intensity was measured using an LSM700 Meta confocal microscope.

(11) The results showed that, when a liver cancer cell line was treated with EGF, compared to a control, microvesicle and TNT formation increases between cells, and this phenomenon is decreased by treatment with the NF-κB inhibitor (WGA, cell membrane staining, FIG. 2A). In addition, in a CD133 expression-stable cell line, it was confirmed that microvesicles and TNT formation rates highly increase (FIG. 2B). Therefore, it was expected that microvesicle budding is closely related to the change in CD133 expression.

(12) In addition, to bud microvesicles from the cell membrane, the regulation of activities of small GTPases such as ARF6, RhoA and Rac1 is known to be important, and thus an expression pattern of the gene according to the CD133 expression pattern was observed by small GTPase pull-down assay. Specifically, to measure Rac1 activity, after transfected with CD133, cells were harvested, and activity was measured using a Rac1 activation Assay kit. To measure RhoA activity, after CD133 transfection, cells were harvested, and activity was measured using a RhoA activation Assay kit. As a result, it was seen that, in the CD133 expression-stable cell line, Rac1 (NCBI Gene ID: 5879) activity decreases, RhoA (NCBI Gene ID: 387) activity increases, and thus the activity of the downstream signaling molecule Erk1/2 (NCBI Gene ID: 5595, 5594) increases (FIG. 2E). This result showed that a certain level of CD133 expression is essential for microvesicle formation, and particularly, small GTPase activity is regulated to promote microvesicle budding.

EXAMPLE 3

Analysis of Physiological Properties of Microvesicles Released From Cancer Cells

(13) Microvesicles were observed by microvesicle microscopic analysis. Specifically, microvesicles were isolated from the culture solution of CD133-transfected cells and stained with WGA-488, general cells were treated with the stained microvesicles, and after 12 hours, fluorescence intensity was measured using a LSM700 Meta confocal microscope.

(14) It was observed that the microvesicles were released from various types of cancer cells, and contained CD133 (FIG. 3A). In addition, the CD133 (red)-containing microvesicles were isolated by centrifugation, and treated with WGA to stain the cell membrane. It was observed that, when the cell line was treated with the microvesicles, the microvesicles were transported to adjacent cells along with CD133 (FIG. 3B).

EXAMPLE 4

Identification of CD133 Role in Colon Cancer

(15) Forty-eight hours after a HCT116 cell line was transfected with a shCD133 vector into which the shRNA sequence (5′: GAGUCGGAAACUGGCAGAUAGCAAU-3′: SEQ ID NO: 3) knocking down the CD133 expression was inserted to prepare a CD133 expression-inhibited cell line, selection was performed with a selection marker Zeocin. Afterward, single colonies were selected and subcultured, and subjected to Western blotting with wild-type HCT116 to verify CD133 knockdown. The used vector is a vector prepared by substituting a neomycin antibiotic region with Zeomycin in a pSilencer 2.1-U6 neo vector (https://www.thermofisher.com/kr/ko/home/life-science/dna-rnapurification-analysis/napamisc/vector-maps/psilencer-2-1-u6-hygro-vectormap.html).

(16) To isolate microvesicles, the culture solutions of a CD133 normal expression cell line and a CD133 expression-inhibited cell line were collected, and then each cell culture solution was centrifuged at 20,000 g for 1 hour, thereby obtaining a supernatant. After discarding the supernatant, microvesicles were isolated.

(17) From the isolated microvesicles, the amount and sizes of microvesicle budding were measured by a Malvern Nanosight Nanoparticle Tracking Analysis system.

(18) KRAS (NCBI Gene ID: 3845) G13D is a well-known oncogenic protein, and usually found as a KRAS mutant in colon cancer. It was observed that, when CD133 expression was inhibited in a colon cancer cell line, KRAS G13D was not contained in the microvesicles (FIG. 4A). In addition, it was confirmed that CD133 expression is involved in determination of the amount and sizes (100 to 200 nm) of microvesicle budding (FIGS. 4B and 4C).

(19) To observe the transport of KRAS G13D to adjacent cells, microvesicles were isolated from the CD133 expression-inhibited cell line and the general cell line and then normal cells were treated with the microvesicles. Specifically, for cell migration assay, microvesicle-recipient cells were cultured in an 8-μm pore insert, treated with microvesicles, and then after 48 hours, migrating cells counted. For invasion assay, an 8-μm pore insert was coated with Matrigel, and then recipient cells were incubated. Forty-eight hours after microvesicle treatment, cells migrating through the Matrigel were counted.

(20) As a result, it was confirmed that, in normal cells treated with the microvesicles isolated from the general cell line, KRAS G13D is transported to adjacent cells via the microvesicles along with CD133, and the activation of KRAS downstream signaling molecules Akt (NCBI Gene ID: 207) and Erk1/2 increases (FIG. 4D). The microvesicle-mediated KRAS G13D transport induced increases in cell migration to (FIG. 4E) and invasiveness (FIG. 4F) of normal cells. Based on this result, it was able to be confirmed that CD133 regulates a microvesicle transport material, and plays a crucial role in microvesicle size and budding.

EXAMPLE 5

Confirmation of Induction of Anticancer Agent Resistance to Adjacent Cells by CD133-Containing Microvesicles in Colon Cancer

(21) While gefitinib is an anticancer material that inhibits EGFR activity and thus controls cancer cells, it was reported that, in certain types of cancer in which EGFR downstream signaling molecules including KRAS are activated, gefitinib efficacy was insignificant. It was inferred that the transport of KRAS G13D to adjacent cells by CD133-containing microvesicles is involved in resistance to such an EGFR-targeting agent.

(22) Cells being cultured to confirm the above inference were incubated with gefitinib and the microvesicles for a suggested time, cell viability was analyzed using an EZ-cytox kit at specific time, and the absorbance was measured at 570 nm using a microplate reader, followed by calculating a cell growth rate. Twenty-four hours after treatment with the microvesicles and gefitinib, the cells were harvested, and then fixed with 95% cold ethanol. The fixed cells were treated with RNase and propidium iodide to stain DNA, and then the stained DNA was analyzed by FACS.

(23) As a result, it was confirmed that, when treated along with gefitinib, microvesicles extracted from CD133 expression-inhibited colon cancer cells do not contribute to cell proliferation of adjacent recipient cells, but microvesicles extracted from CD133-expressing cells restore cell proliferation even when gefitinib is treated (FIGS. 5A and 5B). To demonstrate that such a change is caused by KRAS G13D transport by microvesicles, the change in KRAS downstream signaling molecules and target genes in microvesicle-recipient cells was observed. It was confirmed that the CD133-mediated KRAS G13D transport increases the activities of KRAS downstream signaling molecules FAK (NCBI Gene ID: 5747), Akt and Erk1/2 in the recipient cells (FIG. 5C). In addition, it was observed that mRNA expressions of KRAS target genes SERVIVIN (NCBI Gene ID: 332) and CCNB1 (NCBI Gene ID: 891) increase, anti-apoptotic mRNA (BCLXL, BCL2L2 (NCBI Gene ID: 598, 599)) increases, and pro-apoptotic mRNA (BIM (NCBI Gene ID: 10018)) decreases (FIG. 5D). This shows that the cell migration to and invasiveness of recipient cells receiving the oncogenic protein (KRAS G13D)-containing microvesicles increase, and thus the development of cancer is maintained after treatment with an anticancer agent. In conclusion, in colon cancer, CD133-containing microvesicles are involved in transport of the KRAS oncogenic protein to induce anticancer agent resistance to adjacent cells, thereby accelerating cancer development.