KR101665565B1 - Screening Method for Therapeutic Agent Capable of Treating Cancer Having Resistance to Epidermal Growth Factor Receptor Blocking Agent - Google Patents

Abstract

The present invention relates to an anticancer agent comprising a KITENIN / ErbB4-Dvl2-c-Jun axis signaling pathway activation block as an active ingredient, a method for screening the anticancer agent, a composition for screening the anticancer agent, a therapeutic agent for inhibiting EGFR A method for providing information necessary for determining the reactivity to an epithelial growth factor receptor (EGFR) receptor, and a kit for determining the reactivity against an epithelial growth factor receptor (EGFR) blocking agent. According to the present invention, it is possible to effectively screen candidates for anticancer drugs against cancer that do not show reactivity with the EGFR blocking agent, and the anticancer drug candidates of the present invention can be used for the treatment of cancer patients who do not have anti- It can be used as an adjuvant. In addition, according to the present invention, it is possible to provide important and necessary information for determining whether to use the EGFR blocking agent and the therapeutic prognosis of the EGFR blocking agent.

Description

TECHNICAL FIELD [0001] The present invention relates to a screening method for an anticancer agent capable of treating a cancer having resistance to an epithelial growth factor receptor blocker,

The present invention relates to an anticancer agent comprising a KITENIN / ErbB4-Dvl2-c-Jun axis signaling pathway activation blocker as an active ingredient, a method for screening the anticancer agent, a composition for screening the anticancer agent, And a kit for determining the reactivity against an EGFR blocking agent.

Epidermal Growth Factor (EGF) is secreted from the tumor-associated microenvironment by secretory signaling and plays an important role in promoting metastasis. The EGF receptor (EGFR, ErbB1) is a cell surface receptor for the EGF-family member, and the ErbB family includes four different tyrosine kinases (ErbB1, ErbB2, ErbB3, ErbB4) that are activated after binding to the ErbB ligand. ErbB tyrosine kinase mediates complex interactions between tumor cells and their microenvironment, which can ultimately lead to increased tumor development, and ErbB has thus become an interesting target for therapeutic intervention in some human cancers 1, 2). Although EGFR is over-expressed in CRC (colorectal cancer) tissues, EGFR kinase inhibitors, which have been proven effective in other clinical settings, often fail to block metastatic recurrence of CRC (3, 4). The reason for this reaction is due to the heterogeneity of CRC. Previous studies have shown that CRCs (so-called "quadruple negative" tumors) with no tumorigenesis in the KRAS, BRAF, PIK3CA and PTEN genes have the highest therapeutic potential in anti-EGFR therapies (5, 6) . However, approximately 20% of patients with metastatic CRC who do not respond to anti-EGFR target therapy have no mutations in KRAS, BRAF, and PIK3CA, or no loss of PTEN expression (7). With such low reactivity in these CRC patients, the researchers have begun to work extensively to understand the kinase-independent EGFR function that can support tumor growth. In one such study, it has been reported that EGFR maintains intracellular glucose basal levels irrespective of its kinase activity, thereby preventing the cells from progressing to autophagy even in the presence of EGFR kinase inhibitors and increasing tumor cell survival (8). Therefore, it is very important to identify novel downstream signals of EGF that do not contain EGFR, since such signals impart CRS cells a pro-survival response to anti-EGFR therapy. Nevertheless, these signals have not yet been identified. In previous studies, we have found that KITENIN (KAI1 C-terminal interacting tetraspanin) is involved in the dispersion of colon cancer cells 11, 12, squamous cancer cells 13-15, stomach cancer cells 16 and liver cancer cells 17 Respectively. Expression of the KITENIN protein is higher than that of the corresponding normal mucosal tissue or early cancer tissue in human colon cancer tissues 11 and 12, laryngeal cancer tissue 14, oral squamous cancer tissue 15, gastric cancer tissue 16 and liver cancer tissue 17 ), Respectively. The interaction of KITENIN with Dvl (Disheveled) / PKCδ is important in controlling CRC cell infiltration through ERK / AP-1 activation (11), and KITENIN expression is significantly associated with low lymph node metastasis and low CRC survival 12). However, it is not known how increased KITENIN can induce CRC development and that external signals such as EGF or HGF from the tumor-associated microenvironment can contribute to colorectal tumorigenesis by modulating downstream signaling pathways of tumorigenic KITENIN .

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Korean Patent Publication No. 10-2011-0090842 Korean Patent Publication No. 10-2012-0138226

1. De Luca, et al. J Cell Physiol 2008; 214: 559-67. 2. Huang Z, Brdlik C, Jin P, Shepard HM. Expert Opin Biol Ther 2009; 9: 97-110. 3. Arnold D, Seufferlein T. Gut 2010; 59: 838-58. 4. Bardellia, Siena S. J Clin Oncol 2010; 28: 1254-61. 5. Van Cutsem E, et al. Eur J Cancer 2013; 49: 2476-85. 6. Custodioa, Feliu J. Crit Rev Oncol Hematol 2013; 85: 45-81. 7. Sartore-Bianchia, et al. PLoS One 2009; 4: e7287. 8. Weihua Z, et al. Cancer Cell 2008; 13: 385-93. 9. Lee JH, et al. Cancer Res 2004; 64: 4235-43. 10. Lee JH, et al. Cancer Res 2005; 65: 8993-9003. 11. Kho DH, et al. Gut 2009; 58: 509-19. 12. Lee S, et al. Pathol Int 2011; 61: 210-20. 13. Lee JK, et al. Otolaryngol Head Neck Surg 2010; 142: 747-52. 14. Lee JK, et al. Laryngoscope 2010; 120: 953-8. 15. Yoon TM, et al. Auris Nasus Larynx 2013; 40: 222-6. 16. Ryu HS, et al. Anticancer Res 2010; 30: 3479-86. 17. Cho SB, et al. Oncol Res 2011; 19: 115-23. 18. Li L, et al. J Biol Chem 1999; 274: 129-34. 19. Wiley HS, Walsh BJ, Lund KA. J Biol Chem 1989; 264: 18912-20. 20. Egeblad M, et al. Biochem Biophys Res Commun 2001; 281: 25-31. 21. Davis RJ. Cell 2000; 103: 239-52. 22. Davies CC, et al., Nat Cell Biol 2010; 12: 963-72. 23. Ahmed D, et al. Oncogenesis 2013; 2: e71. 24. Ding W, et al. Proc Natl Acad Sci USA 2008; 105: 13433-8. 25. Li Z, et al., Hepatogastroenterology 2011; 58: 411-6. 26. Yu SY, et al., Chem Biol Interact 2013; 203: 412-22. 27. Bedie, et al., Mol Cancer Ther 2012; 11: 2429-39. 28. Ozanne BW, et al., Oncogene 2007; 26: 1-10. 29. Nateri AS, Nature 2005; 437: 281-85. 30. Bogoyevitch MA, Kobe B. Microbiol Mol Biol Rev 2006; 70: 1061-95. 31. Keshet Y, Seger R. Methods Mol Biol 2010; 661: 3-38. 32. Casaletto JB, McClatchey AI. Nat Rev Cancer 2012; 12: 387-400. 33. Clayton AH, et al. J Biol Chem 2005; 280: 30392-9. 34. Chung I, Nature 2010; 464: 783-7. 35. Wheeler DL, et al., Nat Rev Clin Oncol 2010; 7: 493-507. 36. Valtorta E, et al., Int J Cancer 2013; 133: 1259-65.

We have found that the extracellular signal affects an enhanced KITENIN-induced increase in CRC (colorectal cancer) cell infiltration and that the action of extracellular signals on KITENIN, such as epithelial growth factor (EGF) The aim of this study was to investigate whether patients were associated with failure of anti-EGFR therapy. As a result, we have identified the novel EGFR-independent teratogenic EGF signaling pathway, KITENIN / ErbB4-Dvl2-c-Jun axis, which operates under the coexpression of KITENIN and ErbB4, , And that activation of the pathway serves as a new mechanism for acquiring resistance to EGFR blocking agents regardless of EGFR in colorectal cancer. Thus, the present invention has been completed.

Accordingly, it is an object of the present invention to provide an anticancer agent comprising as an active ingredient a signaling pathway activating block agent of the KITENIN / ErbB4-Dvl2-c-Jun axis.

Another object of the present invention is to provide a method for screening the anticancer agent candidate substance.

It is still another object of the present invention to provide a composition for screening the anticancer drug candidate.

It is yet another object of the present invention to provide a method for providing information necessary for judging whether or not a therapeutic agent for blocking epithelial cell growth factor receptor (EGFR) is reactive.

It is still another object of the present invention to provide a kit for determining the reactivity to an EGFR blocking agent.

The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a method of screening an anticancer drug candidate comprising the steps of:

(a) treatment of candidate substances with the treatment of epidermal growth factor (EGF) with cells having a signaling pathway of KITENIN / ErbB4-Dvl2-c-Jun axis, step;

(b) measuring the degree of activation of the signal transduction pathway of the KITENIN / ErbB4-Dvl2-c-Jun axis in the treated cells; And

(c) When activation of the signaling pathway of the KITENIN / ErbB4-Dvl2-c-Jun axis is further inhibited in the cells treated with the candidate substance compared to the cells not treated with the candidate substance, the candidate substance is selected as an anticancer drug candidate Judging step.

Epidermal growth factor (EFG) binds to the epidermal growth factor receptor (EGFR) to increase the invasion of cancer cells, thereby promoting cancer progression. Based on this, monoclonal antibodies against EGFR - kinase inhibitor or EGFR are used for the treatment of colorectal cancer. However, even when a therapeutic agent such as Cetuximab or Erbitux is administered as an EGFR blocking agent, for example, as a monoclonal antibody against EGFR in the treatment of metastatic colorectal cancer, the treatment effect in 20% (Non-reactivity) is reported.

The inventors have for the first time established that EGF activates the novel signaling pathway of the KITENIN / ErbB4-Dvl2-c-Jun axis independently of the signal pathway by EGFR, thereby further increasing the invasion of colon cancer cells. According to the experimental results of the following specific examples of the present invention, in patients with stage 4 metastatic colorectal cancer treated with monoclonal antibody against EGFR, patients with good initial treatment response were treated with KITENIN staining Respectively. These results suggest that the activation of KITENIN / ErbB4 - Dvl2 - c - Jun by EGF acts as a novel mechanism of resistance to EGFR block therapy independently of EGFR in colorectal cancer.

Based on the facts proven in one embodiment of the present invention, searching for an active substance that blocks EGF-induced KITENIN / ErbB4-Dvl2-c-Jun axis signaling pathway activation, New anticancer drug candidates effective against cancer can be selected.

According to a preferred embodiment of the present invention, the cancer to be treated in the anticancer drug candidate screening method is a cancer which shows no reactivity with the epithelial growth factor receptor (EGFR) blocking agent.

According to a preferred embodiment of the present invention, the cancer to be screened is a breast cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or ocular melanoma, uterine sarcoma, ovarian cancer, Cancer, prostate cancer, bronchial cancer, or bone marrow cancer, and more preferably, the cancer is cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, small bowel cancer, endocrine cancer, thyroid cancer, pituitary cancer, kidney cancer, soft tissue tumor, Colon cancer or lung cancer.

In the present invention, the anticancer drug candidate is a substance that inhibits the expression of a protein that constitutes the signal transduction pathway. For example, RNAi (RNA interference) molecules, antisense oligonucleotides, siRNA, shRNA, Or miRNA. ≪ / RTI >

In the present invention, the anticancer drug candidate is a substance that binds to a protein constituting the signal transduction pathway and inhibits its activity or its interaction with other proteins. For example, an oligopeptide, a monoclonal antibody, a polyclonal antibody, But are not limited to, a chimeric antibody, a humanized antibody, a peptide nucleic acid (PNA), an aptamer, a low molecular weight or a high molecular compound.

Compounds that can be used as candidates in the present invention can be obtained from libraries of synthetic or natural product compounds and synthetic compound libraries are available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA) And Sigma-Aldrich (USA), and libraries of natural compounds are commercially available from Pan Laboratories (USA) and MycoSearch (USA). In addition, the compounds may be obtained by various combinatorial library methods known in the art.

According to another preferred embodiment of the present invention, the inhibition of activation of the signal transduction pathway of the KITENIN / ErbB4-Dvl2-c-Jun axis in step (c) of the present invention is (i) inhibition of binding of KITENIN to ErbB4, Ii) inhibition of Dvl2 degradation in combination with KITENIN, (iii) inhibition of increased c-Jun protein content, or (iv) inhibition of AP-1 transcription factor activation.

According to another aspect of the invention, epithelial growth factor (EGF); And KITENIN / ErbB4-Dvl2-c-Jun axis.

According to a preferred embodiment of the present invention, in the composition for screening an anticancer agent, the subject cancer is a cancer which shows no reactivity to an EGFR blocking agent.

According to a preferred embodiment of the present invention, the cancer to be treated of the anticancer agent is selected from breast cancer, lung cancer, gastric cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or ocular melanoma, uterine sarcoma, Cancer of the endometrium, cancer of the cervix, cancer of the endometrium, endocrine cancer, thyroid cancer, parathyroid cancer, renal cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchus cancer or bone cancer, Is colon cancer or lung cancer.

According to another aspect of the present invention, there is provided an anticancer agent comprising, as an active ingredient, an activating blocking agent for the signal transduction pathway of the KITENIN / ErbB4-Dvl2-c-Jun axis.

The activator of the KITENIN / ErbB4-Dvl2-c-Jun axis signaling pathway may be a substance that inhibits the expression or activity of the component protein of the signal transduction pathway.

In the present invention, the activation blocking agent is a substance that inhibits the expression of a protein constituting the signal transduction pathway. The RNAi (RNA interference) molecule, the antisense oligonucleotide, the siRNA, the shRNA, or the miRNA But is not limited thereto.

In the present invention, the activating blocking agent is a substance which binds to the protein constituting the signal transduction pathway and inhibits its activity or interaction with other proteins, and includes oligopeptides, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, But are not limited to, humanized antibodies, peptide nucleic acids (PNA), aptamers, low molecular weight or high molecular compounds.

According to a preferred embodiment of the present invention, the activating block of the signaling pathway of the KITENIN / ErbB4-Dvl2-c-Jun axis includes (i) inhibition of binding of KITENIN to ErbB4, (ii) (Iii) inhibition of an increase in the amount of c-Jun protein, or (iv) inhibition of AP-1 transcription factor activation.

According to a more preferred embodiment of the present invention, the activation blocking agent may be a substance that specifically binds to the KITENIN or ErbB4 protein to inhibit binding of the two proteins.

According to a preferred embodiment of the present invention, the cancer to be treated of the anticancer agent is selected from breast cancer, lung cancer, gastric cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or ocular melanoma, uterine sarcoma, Cancer of the endometrium, cancer of the cervix, cancer of the endometrium, endocrine cancer, thyroid cancer, parathyroid cancer, renal cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchus cancer or bone cancer, Is colon cancer or lung cancer.

According to a preferred embodiment of the present invention, the cancer to be treated of the anticancer agent is a cancer showing no reactivity to the EGFR blocking agent.

According to another aspect of the present invention, there is provided a method for determining the reactivity of an EGFR blocking agent or an EGFR blocking agent comprising measuring the activation level of a signal transduction pathway of KITENIN / ErbB4-Dvl2-c- And provides a method for providing information necessary for judging the prognosis of treatment.

In cancer patients, EGFR blocking agents are administered only when the gene is normal, by examining the mutation of genes such as K-Ras and B-Raf in cancer tissues of patients who have been excised before using EGFR blocking agents. Although the initial treatment effect is observed in most patients, the initial treatment effect does not appear in some patients even though the K-Ras or B-Raf gene is normal.

Therefore, additional markers are needed to determine whether to use EGFR blocking agents in patients after they are identified as cancer.

According to the results of the present invention, activation of the signaling pathway of KITENIN / ErbB4-Dvl2-c-Jun axis as a distinct signal transduction pathway independent of EGFR in cancer cells is highly correlated with non-reactivity to EGFR blocking agents do. Therefore, the activation of the signaling pathway of KITENIN / ErbB4-Dvl2-c-Jun axis was examined in cancer tissues extracted before using EGFR blocking agent in metastatic cancer patients, And to provide information that is very important and necessary to determine the prognosis of the disease.

According to a preferred embodiment of the present invention, measuring the degree of activation of the signaling pathway of the KITENIN / ErbB4-Dvl2-c-Jun axis comprises measuring the amount of KITENIN protein.

According to a preferred embodiment of the present invention, the cancer is selected from the group consisting of breast cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or ocular melanoma, uterine sarcoma, ovarian cancer, Cancer, endometrial cancer, endometrial cancer, small intestine cancer, endocrine cancer, thyroid cancer, pituitary cancer, kidney cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchial cancer, or bone cancer.

According to another aspect of the present invention, there is provided a kit for determining the reactivity of a cancer containing a KITENIN protein detection reagent to an epithelial growth factor receptor (EGFR) blocking agent.

According to a preferred embodiment of the present invention, the cancer in the kit is selected from the group consisting of breast cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or ocular melanoma, uterine sarcoma, Cancer, endometrial cancer, endometrial cancer, small intestine cancer, endocrine cancer, thyroid cancer, pituitary cancer, kidney cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchial cancer, or bone cancer.

The features and advantages of the present invention are summarized as follows:

(i) According to the present invention, screening for a substance blocking activation of the signal transduction pathway of the KITENIN / ErbB4-Dvl2-c-Jun axis can effectively screen candidate anticancer agents against cancer that does not show reactivity with the EGFR blocking agent .

(Ii) The anticancer drug candidate of the present invention can be used as an anti-cancer adjuvant in the treatment of cancer patients who do not have anticancer effect by the EGFR blocking agent.

(Iii) In patients with metastatic cancer, activation of the signaling pathway of KITENIN / ErbB4 - Dvl2 - c - Jun axis was investigated in cancer tissues extracted before using EGFR blocking agent. Whether to use EGFR blocking agent And may provide important and necessary information in determining the prognosis of treatment with EGFR blocking agents.

Figure 1 shows that in KITENIN-transfected cells, EGF upregulates AP-1 and enhances cell migration potential.
Panel A of Figure 1 shows the effect of growth factors on AP-1 activity under KITENIN-transfection. 293T cells were co-transfected with KITENIN as an AP-1 reporter or without KITENIN or with KITENIN siRNA and treated with EGF (100 ng / ml), HGF (40 ng / ml), or 10% FBS for 12 hours Respectively. Each bar represents the mean ± SEM for three samples. si-scr, non-specifically scrambled siRNA (negative control). @, Significant difference between groups (@ P <0.05).
Panel B of Fig. 1 shows the results of infiltration analysis in Caco2 cells after EGF treatment. Caco2 / vector or Caco2 cells stably transfected with KITENIN (Caco2 / KITENIN) were subjected to infiltration analysis after treatment with EGF (100 ng / ml) for 12 hours. The figure shows three independent experiments. The histogram represents infiltrating cells counted in five selected areas and is shown as a bar graph (mean ± SEM, n = 3). An asterisk (* or @) represents a significant difference between the different groups (*** P <0.001, @@ P <0.01, @@@ P <0.001).
Panel C in Fig. 1 shows the results of cell growth analysis in Caco2 cells after EGF treatment. Anchorage independent cell growth assays were performed on Caco2 / vector or Caco2 / KITENIN cells. EGF (100 ng / ml) was treated for 12 hours. The figure shows three independent experiments. The area of stained cells was measured and displayed as a bar graph (mean ± SEM, n = 3).
Figures 2A-2D show that EGF exhibits synergistic AP-1 activation and enhanced CRC cell migration capability under enhanced KITENIN conditions in an EGFR independent manner.
FIG. 2A shows the results of measuring the effect of EGFR-knockdown on AP-1 synergy and increased cell infiltration by EGF. Caco2 / vector or Caco2 / KITENIN cells treated with EGF for 12 hours were subjected to AP-1 reporter analysis (left panel) and infiltration analysis (right panel). NS, no significant difference between groups.
Figure 2B shows the effect of chemical blockade of EGFR on increased cellular infiltration by EGF. Caco2 / vector or Caco2 / KITENIN cells were treated with EGF and / or AG1478 (1 [mu] M) for 12 hours.
Figures 2C and 2D show the action of EGF in MEF and SW-620 cells that do not express EGFR. Figure 2C shows the results of transfection of EGFR-wild type (upper panel) and EGFR-knockout (lower panel) MEF cells with KITENIN and treatment with EGF for 1 hour. 2D, SW-620 cells (SW-620 / KITENIN) stably transfected with SW-620 / vector or KITENIN were treated with EGF for 12 hours and subjected to anchorage-independent cell growth analysis. Caco2 / vector or Caco2 / KITENIN cells were treated with EGF and / or AG1478 (1 [mu] M) for 12 hours.
Figures 3A-3E show that KITENIN induces an increase in the interaction with Dvl2 in the cytoplasmic ER as well as downregulation of KITENIN-coupled phospho-Dvl2 and upregulation of c-Jun.
Figure 3A shows the effect of PMA on c-Jun and Dvl2 in KITENIN-transfected cells. Immunoreactive Dvl2 / c-Jun was detected after treatment with PMA (100 nM) for 4 hours in 293T cells transfected or not transfected with KITENIN and the results of three experiments were measured with a density analyzer. Phospho-Dvl2 was detected by phosphorylation-dependent mobility shift using Dvl2-specific antibodies. Arrows and arrowheads indicate phospho-Dvl2 and unphospho-Dvl2, respectively.
Figure 3B shows the results of measuring the effect of EGF on c-Jun and Dvl2 (upper panel) and KITENIN-bound phospho-Dvl2 (lower panel) in CRC cells. Serum depleted HCT116 cells were treated with EGF at designated time points. Total cell lysates (500 μg) were treated with or without lambda phosphatase (lambda Ppase) and analyzed by immunoblot (upper panel) or immunoprecipitation (lower panel). Arrows and arrowheads indicate phospho-Dvl2 and unphospho-Dvl2, respectively. The upper band disappeared after the phosphatase treatment.
Figure 3C shows the effect of transfection with Dvl2 or DN-c-Jun on increased KITENIN conditions on EGF-induced AP-1 synergy. 293T cells were co-transfected with Dvl2 or DN-c-Jun with AP-1 reporter and KITENIN or without KITENIN and treated with EGF or PMA (20 nM) for 12 hours.
FIG. 3D is the result of measuring the effect of EGF on the coexistence of Dvl2 and KITENIN in cytoplasmic ER. 293T cells (upper panel) were transiently transfected with Dvl2-GFP and KITENIN-HA constructs, and HCT116 cells (lower panel) were transiently transfected with Dvl2-GFP for 36 hours. Cells were visualized with a fluorescent confocal microscope after treatment with EGF for 30 min. A representative example is as follows: In an integrated panel, yellow indicates co-existence of Dvl2 with KITENIN.
Figure 3E shows the effect of dvl2 phosphorylation regulation on c-Jun (left panel) and AP-1 synergy (right panel) by EGF. 293T cells were treated with casein kinase I inhibitor D4476 (100 [mu] M) for 4 hours to block Dvl phosphorylation, after which Dvl2, c-Jun, and c-Fos levels were measured (left panel). Next, cells were co-transfected with AP-I reporter and casein kinase I e with or without KITENIN and then treated with EGF or PMA (20 nM) for 12 h (right panel).
Figure 4 shows that ErbB4 is required for AP-1 synergy by EGF under increased KITENIN conditions.
Panel A of FIG. 4 shows the effect of RON-knockdown or ErbB4-knockdown on increased cell infiltration by KITENIN. Caco2 / vector or Caco2 / KITENIN cells were transfected with si-scr, si-RON, or si-ErbB4 for 48 hours and infiltration analysis was performed.
Panel B of FIG. 4 shows that EGF increases KITENIN's interaction with ErbB4. Caco2 / KITENIN cells were treated with EGF for 12 hours and immunoreactive with KITENIN and ErbB4 were immunoprecipitated with anti-ErbB4 antibody.
Panel C of Figure 4 shows that ErbB4 is required for EGFKITENIN-AP-1 signaling. 293T cells were co-transfected with ErbB4-siRNA treatment or KITENIN and AP-1 reporter without this treatment, and then treated with EGF for 12 hours.
Panel D of FIG. 4 shows that the interaction between KITENIN and Dvl2 was reduced by ErbB4-knockdown (upper panel) and restored EGF action under increased KITENIN conditions (lower panel). Caco2 / KITENIN cells were transfected with Dvl2 and treated with ErbB4-siRNA for 36 hours. Immunoreactive Dvl2 or KITENIN was tested after immunoprecipitation with an anti-KITENIN or anti-Dvl2 antibody, respectively (top panel). Caco2 / KITENIN cells were treated with ErbB1-siRNA or ErbB4-siRNA for 36 hours. Immunoreactive Dvl2 and c-Jun were examined after EGF treatment (bottom panel).
Figures 5A-5D show that the EGF-KITENIN / ErbB4-c-Jun axis upregulates CRC cell infiltration.
Figure 5A shows that the EGFKITENIN / ErbB4-c-Jun axis works in CHO cells. CHO-K1 cells were co-transfected with ErbB4 JM-a / CYT-2 (a2) or KITENIN without ErbB4 JM-a / CYT-2 (a2) for 48 hours and treated with EGF for 12 hours.
FIG. 5B shows the results of infiltration analysis in ErbB4-transfected HCT116 cells after EGF treatment with or without KITENIN-knockdown. HCT116 cells were transfected with ErbB4 isoform (a2) or co-transfected with si-KITENIN / ErbB4 isoform (a2). Cells or transfected HCT116 cells were treated with EGF for 12 hours and infiltration assays were performed.
Figure 5C shows the effect of ErbB kinase-blocking agents on increased cellular infiltration by EGF under KITENIN / ErbB4 conditions. Caco2 / ErbB4 (a2) or Caco2 / KITENIN / ErbB4 (a2) cells were treated with EGF and / or AG1478 (1 μM) for 12 hours and infiltration assays were performed.
Figure 5D schematically illustrates a novel downstream signaling pathway of apoptotic EGF: stimulating the KITENIN / ErbB4-Dvl2-c-Jun axis by EGF upregulates c-Jun and leads to CRC cell infiltration. Unidentified routes are indicated by dashed lines.
Figure 6 shows that KITENIN / ErbB4 is coexpressed at high levels in tumor tissue from metastatic CRC patients with poor response to cetuximab treatment. Panel A of FIG. 6 shows the results of immunohistochemistry (dark brown) of the expression of KITENIN and ErbB4 in a continuous flap of colon tissue obtained from metastatic CRC patients with wild-type KRAS treated with cetuximab. Representative examples are: # 1, # 2, and # 3 are CRC patients with stage IV at 2 months post-treatment with cetuximab. # 4 and # 5 are patients who showed PD for the same period. Scale bar: 50 μm.
Panel B of FIG. 6 shows a positive correlation between the KITENIN expression score and poor response to cetuximab in the early stages after cetuximab treatment. To assess the immunohistochemical levels of KITENIN and ErbB4, the scores were calculated and the significance between PR and PD groups tested. NS, not significant.
Figure 7 shows that EGF induces synergistic AP-1 activity under increased KITENIN in an EGFR- and EGFR-kinase independent manner. Panel A of Figure 7 shows the effect of EGFR-knockdown on AP-1 by EGF. Panel B of Figure 7 shows the effect of chemical blockers of EGFR on AP-1 by EGF.
Figure 8 shows the effect of functional blockade of EGFR through anti-EGFR monoclonal antibody treatment on increased cell infiltration by EGF. In panel A of FIG. 8, Caco2 cells were treated with EGF (100 ng / ml) and an increased dose of cetuximab (1, 3, 10 μg / ml) for 12 hours. In panel B of FIG. 8, Caco2 / vector or Caco2 / KITENIN cells were treated with EGF and / or cetuximab (10 μg / ml) for 12 hours and infiltration analysis was performed.
Figure 9 shows that under increased KITENIN conditions, EGF-induced AP-1 synergy does not require activated JNK. Panel A of Figure 9 shows the effect of EGF on phospho- / total-JNK and phospho / total-c-Jun. Panel B of Figure 9 shows the effect of JNK blockers on EGF action under increased KITENIN conditions.
Figure 10 shows the effect of Dvl2-knockdown on c-Jun and the effect of EGF on c-Jun / Dvl2 levels in KITENIN transfected cells. In panel A of FIG. 10, 293T cells were transfected for 48 hours with increasing amounts of Dvl2 or Dvl2 siRNA, and AP-1 or TOPflash reporter activity and c-Jun levels were measured. (Panel B in Figure 10) and Caco2 cells (Panel C in Figure 10) without transfection or transfection of immunoreactive Dvl2 and c-Jun with KITENIN in Panel B-C of Figure 10 for 8 hours Were examined after EGF treatment and measured by density meter. The experiment was repeated 3 times.
Figure 11 shows that Dvl2-knockdown does not increase transcription of c-Jun and Dvl2 does not interact with c-Jun. Panel A of FIG. 11 shows the effect of Dvl2 knockdown on c-Jun promoter activity. Panel B in Figure 11 shows that Dvl2 does not interact directly with c-Jun.
Figure 12 shows the modulation of multiple RTKs in KITENIN-overexpressed Caco2 or KITENIN-knockdown HCT116 CRC cells. Panel A of Figure 12 shows that ErbB4 and RON are positively regulated by KITENIN. Panel B of FIG. 12 shows that phospho-ErbB4 and phospho-RON are regulated by KITENIN. Panel C of Figure 12 shows that KITENIN interacts with ErbB4.
Figure 13 shows that cotransfection of KITENIN and ErbB4 induces further increased AP-1 activity after EGF treatment.
Figure 14 shows that KITENIN high-affinity affects the survival rate of CRC cells on the antiproliferative effect of cetuximab. Panel A of Figure 14 shows the sensitivity of various CRC cells to cetuximab. Panel B of Figure 14 shows the expression level of KITENIN and the survival rate for Cetuximab of KRAS / BRAF wild type Caco2 CRC cells. Panel C of FIG. 14 shows the expression level of KITENIN and the survival rate of corteximab of the KRAS / BRAF mutant cell lines DLD1 and HCT116 CRC cells.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

1. Experimental methods and materials

Plasmids and cell lines

The expression constructs of HA- or V5-tagging KITENIN, GST-tagging KITENIN truncation mutants, HA-tagging ErbB4 truncation mutants, EGFR, HA-tagging Dvl2, DN-cjun and Nrdp1 were constructed by PCR-based methods. All constructs were identified by sequencing. ErbB4 cDNA was provided by K. Elenius (University of Turku, Finland). Human CRC cell lines (Caco2, DLD-1, HCT116, HT29, SW-480 and SW-620), CHO-K1 cells and human embryonic kidney (293T) cells were purchased from the American Type Culture Collection, And cultured (11). The EGFR curing type and knockout MEF were obtained from Z. Dong (University of Minnesota, USA). Construct and si-RNA were transfected into cells using FuGENE6 and RNAiMax, respectively (11). Immunoblotting and immunoprecipitation, siRNA interference and RT-PCR analysis, reporter and infiltration assays, immunofluorescence and immunohistochemistry methods for whether EGF regulates the downstream signaling pathway of tumor positive KITENIN in CRC cells Respectively.

Growth Factors

Human recombinant EGF (R & D systems) and HGF (Invitrogen) were used at a concentration of 40 - 100 ng / ml. In all experiments, cells were placed on overnight serum deprivation conditions and stimulated with appropriate growth factors in 1% serum medium.

Reporter assay

For reporter analysis, 293T cells were plated at a concentration of 5 x 10 ⁴ cells / well at 24 h prior to transfection, followed by the addition of 50 ng of reporter (TOPFLASH, AP-1-luc, and c- luc promoter), 1 ng of phRL-CMV, and an effect construct (50-200 ng) were transiently transfected using FuGENE6. Blank vector DNA was added to make the total amount of DNA equal. After 24 hours or 48 hours of incubation, luciferase activity was measured and normalized for Renilla activity for transfection efficiency (11). All data were expressed as mean ± SEM for three replicate samples.

Antibodies and Immunoprecipitation

ErbB1, ErbB4, c-Met, RON, and Nrdp1 (Santa Cruz Biotech); antibodies to c-Jun, c-fos, p-JNK, total-JNK, p-ERK, p-ErbB4, Dvl2, and beta -catenin; Anti-V5 and anti-myc antibodies (MBL); Anti-HA and anti-actin antibodies (Sigma); And rabbit KITENIN (9) or Vangl1 (Atlas) were used with a suitable secondary antibody (Amersham biosciences). For transient transfection analysis, 293T cells were transfected with various plasmids and recovered for immunoblot analysis at 36 hours post transfection. In most assays using stable cell lines, mixed polyclonal cells were used to exclude clonal mutations. Cell proteins were isolated, transferred, and immunoblotted (9). The blot was re-probed with anti-actin antibody to control loading. Cell lysates from 293T, Caco2, and HCT116 cells were used for immunoprecipitation experiments (11).

Cell invasion assay

Cell infiltration analysis was performed using a Transwell migration apparatus (10). Briefly, cultured cells or stably overexpressing cells treated with siRNA for 48 hours were loaded on top of a 24-well infiltrated chamber assay plate (Costar). Modified DMEM medium containing 20 μg / ml of fibronectin (Calbiochem) as a chemoattractant was added to the lower chamber. After incubation for 24 hours, cells were stained. The cells on the upper surface of the filter are wiped off with cotton balls and the transferred cells on the lower surface

The number of cells was measured in six 0.5 mm x 0.5 mm random squares per filter. Experimental results were expressed as mean ± SEM of cells per field for at least 3 independent experiments.

Analysis of soft agar colony formation

Caco2 (1 x 10 ⁵ ) and SW-620 (1 x 10 ⁵ ) cells were suspended in 1.5 ml of soft agar (0.35% agarose in DMEM complete medium) and 1.5 ml of solidified agar (0.6% agarose in DMEM complete medium), and cultured for 3 and 2 weeks, respectively. Cells were fed with cell culture medium containing EGF or no EGF twice per week. The pixel intensity of the colony region was measured using IMT iSolution software (IMT i-Solution Inc) from a random microscope view for each plate. To measure the% area of the colony, the number of pixels in the colony area was normalized by the given pixel * pixel rectangle. Data were expressed as the mean of three experiments.

Cell viability assay

The method for measuring the growth and survival of CRC cells was performed based on the colorimetric determination of MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] assay. Briefly, CRC cells were plated in 96-well plates (5 x 10 ³ cells / well) and cultured. Cells were treated with different concentrations of cetuximab (0, 10, 30, 100 μg / ml) for 96 h. After incubation, analysis was performed according to the previously described method (9).

Immunofluorescence

Cells were inoculated on a cell culture slide (Becton Dickinson) coated with fibronectin. 293T cells were transfected with an expression vector encoding Dvl-GFP and HA-tagged KITENIN and HCT116 cells transfected with an expression vector encoding Dvl-GFP. After 24 hours, the cells were treated with EGF (100 ng / ml) for 30 minutes under serum depletion conditions. After incubation, monolayer cells were washed, fixed, permeabilized, stained with monoclonal anti-HA antibody coupled to red fluorescently labeled Alexa-fluor 568 secondary antibody, or blue fluorescent nuclei stained with DAPI . Cells were examined with a Laser Scanning Confocal Microscope (Leica) (11).

RT-PCR and RNA interference

RNA preparation and reverse transcription were performed according to previously described methods (11). The RT-PCR logarithm was adjusted to 30 cycles to allow quantitative comparisons of various cDNAs from the same reaction on the GeneAmp PCR system (Eppendorf). For siRNA interference experiments, siRNAs specific for human KITENIN, Dvl2, ErbB1, ErbB4, and RON were designed. For nonspecific scrambled siRNA (si-scr), All Stars Negative Control siRNA (Qiagen) was used. Prepare the double-stranded siRNA transfection and was performed according to the protocol provided by Lipofectamine ^TM RNAiMAX. Cells were treated for 48-72 hours for maximal knockdown and used for Western blotting analysis or infiltration analysis after RT-PCR. PCR primers and siRNA sequences are listed in Table 1 below.

RT-PCR KITENIN Forward: 5'-CTACAATGTAGATGGCCCC-3 '(SEQ ID NO: 1)
Reverse: 5'-AGCCTCATCACTGACAAGCC-3 '(SEQ ID NO: 2) EGFR Forward: 5'-TGGAAAACCTGCAGATCATC-3 '(SEQ ID NO: 3)
Reverse: 5'-TTGCTGAGAAAGTCACTGCT-3 '(SEQ ID NO: 4) mErbB4-JM Forward: 5'-CAGTGTGAGAAAATGGAAGATGGACTCCTC-3 '(SEQ ID NO: 5)
Reverse: 5'-GCACGTTTCTTTTTGATGCTCTTTCTTCTG-3 '(SEQ ID NO: 6) mErbB4-CYT Forward: 5'-AAGAATGGCTAGAGACCCTCA-3 '(SEQ ID NO: 7)
Reverse: 5'-GGACCGCTGGAAGCACTGTGATA-3 '(SEQ ID NO: 8) Genomic-PCR for KITENIN Primer 1 5'-CGACGGCCACTGCTCTCACAT-3 '(SEQ ID NO: 9) Primer 2 5'-ATGACAACTGGGGAGAGACACCA-3 '(SEQ ID NO: 10) Primer 3 5'-CAGCTCCTCCCTCCACAGGA-3 '(SEQ ID NO: 11) siRNA Human KITENIN 5'-GCUUGGACUUCAGCCUCGUAGUCAA-3 '(SEQ ID NO: 12) Human Dvl2 5'-AACUUUGAGAACAUGAGCAAC-3 '(SEQ ID NO: 13) Human ErbB1 5'-UGAUCUGUCACCACAUAAUUACGGG-3 '(SEQ ID NO: 14) Human ErbB4 5'-UAUAGAUGUUUCCUGCGCUGAUUUC-3 '(SEQ ID NO: 15) Human RON 5'-UGAAAGAGAAGCCUCUCAGCACGGA-3 '(SEQ ID NO: 16)

Patient and Immunohistochemistry

From December 2011 to January 2007, the first line formulations at Chonnam National University Hwasun Hospital and Yonsei University Hospital were treated with cetuximab plus irinotecan, fluorouracil and leucovorin (FOLFIRI), or cetuximab plus oxaliplatin, fluorouracil , And 55 cases of unresectable recurrent or metastatic colorectal cancer treated with leukopherin (FOLFOX-4) chemotherapy. The paraffin wax-sulphated tumor tissue used for diagnosis was accessible for all included cases. Patients were divided into colon cancer stages by combining colonoscopy, computed tomographic scans of chest and abdomen, and bone scan of positron emission tomography when clinically indicated. Data on patient demographics, chemotherapy regimens, chemotherapy response, progression-free survival (PFS), and overall survival (OS) were obtained through medical records. The tissue flakes were paraffin-free, rehydrated, washed, stained with KITENIN and ErbB4, and tested according to the already known content (9).

Evaluation of KITENIN and ErbB4 staining in CRC samples

To quantify the immunohistochemical KITENIN and ErbB4 expression, the intensity of the staining intensity was multiplied by the size of the positively stained region according to the previously described method to yield a score (11). Dark brown cell staining intensity was graded as follows: 0, no staining; 1+, weak staining; 2+, intermediate staining; 3+, distinct staining. The staining area was evaluated according to the following criteria: 0, 5% positive staining of cells; 1+, 5% - 25% positive staining; 2+, 25% - 50% positive staining; 3+, &gt; 50% positive staining. The combined score had a maximum of 9 and a minimum of 0. In general, samples with a score> 4 were considered positive for candidate protein expression, but these criteria were not used in this study.

Chemotherapy

A total of 31 patients received cetuximab-FOLFIRI chemotherapy and 21 patients received cetuximab-FOLFOX-4 chemotherapy. Patients in the FOLFIRI group received irinotecan injection for 30- to 90-minute doses at a dose of 180 mg / m ² for 1 day followed by 120 minutes of leucovorin injection at a dose of 200 mg / m ^{2 and} a dose of 400 mg / m ² Borus infusion was continuously injected at a dose of 2400 mg / m &lt; ² &gt; for 1 day and 2 days. FOLFOX-4 group of patients 120 minutes of oxaliplatin (oxaliplatin) in the 85 mg / m ² dose in a day injection, 200 mg / m ² to the injection of leucovorin 120 minutes in a capacity and then, 400 mg / m ² of a bolus in uracil and were subjected to 1 and 600 mg / m ² infusion for 22 hours in two days fluoro. Cetuximab was infused for 60 minutes with cetuximab at a dose of 250 mg / m ² , once per week followed by a 120-minute initial infusion of 400 mg / m ² per day. FOLFIRI or FOLFOX was given after cetuximab infusion on day 1 for each period. Each treatment regimen was repeated every two weeks. Treatment schedules were repeated until disease progression, lack of clinical benefit, unacceptable toxicity, or patient rejection. Hematologic and non hematologic adverse events were assessed. Management of the adverse situation and reduction of the chemotherapeutic agent were carried out in a conventional manner.

Response evaluation

Clinical tumor reactivity was assessed radiographically by CT every 8 weeks according to Response Evaluation Criteria in Solid Tumors (ver. 1.0) (37). PFS is defined as the period from the time of chemotherapy to the development of the disease or from any cause to death. If no circumstances occurred until the final record, the patient was removed at that time. OS was calculated from the point of chemotherapy to death for any reason.

Statistical analysis

Experimental differences were tested using ANOVA and Tukey HSD post hoc test or Student's t test. All statistical tests were performed on a two-sided basis and P-values less than 0.05 were considered statistically significant. Statistical analysis was performed using PASW Statistics 20 (SPSS, an IBM Company, Chicago, IL) software.

2. Experimental results

EGF induced synergistic AP-1 activation and enhanced cell migration ability without EGFR or activated JNK in KITENIN transfected cells.

We have found that transfection of KITENIN in 293T cells results in a 3-fold increase in AP-1 activity, mediated by the interaction of Dvl / PKCδ (11). However, although Dvl is required for JNK / AP-1 activation to move into the plasma membrane, it is not clear how Dvl converts the downstream signal for AP-1 activation (18). In order to investigate whether extracellular signal also affects AP-1 activation in KITENIN-transfected cells, growth factors that positively regulated CRC development such as EGF and HGF as candidate substances were selected. Within 293T cells, treatment with EGF, HGF, and FBS increased AP-1 activity (panel A of FIG. 1). Interestingly, when treated with KITENIN-transfected cells without HGF or FBS, a significant increase in AP-1 activity was observed compared to 293T / parent cells, suggesting that EGF specifically sensitizes KITENIN-overexpressing cells Lt; RTI ID = 0.0 &gt; AP-1 &lt; / RTI &gt; activity. However, the increase in AP-1 activity by EGF alone was attenuated by KITENIN-knockdown. (FIG. 1, panel B) and anchorage-independent cell growth (panel C in FIG. 1) after C-EAC treatment in KITENIN-transfected Caco2 cells in Caco2 CRC cells was significantly Respectively. Thus, it was questioned whether EGFR is associated with increased AP-1 activity and increased CRC cell migration observed after EGF treatment under KITENIN transfected conditions. EGFR-knockdown did not affect the increase in AP-1 activity by EGF in KITENIN-transfected 293T cells (FIG. 7A) or KITENIN transfected Caco2 cells (FIG. 2A, left panel) (Fig. 2A, right panel). &Lt; RTI ID = 0.0 &gt; Interestingly, the level of phospho-ERK after EGF treatment was observed to be approximately the same between the parental cells and the KITENIN-transfected cells. Although the increase in phospho-ERK by EGF treatment was not affected by EGFR-knockdown (Fig. 7A and Fig. 2A), it was explained by report 19 that maximal EGF signaling could be achieved with only 5% receptor occupancy Phospho-ERK does not appear to contribute to the increase in AP-1 activity by EGF under KITENIN-transfection. In addition, even when EGFR is chemically blocked through treatment with ErbB kinase activity inhibitor AG1478 (20) or functionally blocked with EGFP by treatment with anti-EGFR monoclonal antibody cetuximab (4), KITENIN trans- (FIG. 7B) or cell infiltration (FIG. 2B and FIG. 8A and FIG. 8B) by EGF treatment in the cultured cells. Thus, phospho-EGFR did not appear to contribute to the increase in AP-1 activity by EGR under KITENIN-transfected conditions.

EGFR-knockout mouse embryonic fibroblasts (MEF) and EGFR-deleted CRC cells were used to determine whether EGF induces AP-1 activation in an EGFR-independent manner under KITENIN-transfection. However, AP-1 activity was scarcely detected in EGFR-knockout MEF cells and EGFR-disrupted SW-620 cells, presumably due to differences in AP-1 reporter expression; Thus, c-Jun was tested instead of AP-1 after EGF treatment. As a positive control, c-Jun was increased after EGF treatment in the EGFR-wild type (124% density measurement, Figure 2C, top panel, second column), but not in EGFR- knockout MEF cells (100% 2C, bottom panel, second column). It was also observed that c-Jun was significantly increased in KITENIN-transfected EGFR-wild-type MEF cells (138% vs 124%, FIG. 2C, top panel) than in co-vector transfected cells after EGF treatment. However, in EGFR knockout MEF cells, c-Jun increased only in KITENIN-transfected cells after EGF treatment (138% vs 100%, Fig. 2C, bottom panel). EGFR-deficient CRC cell line SW-620 cells were used to examine EGF action. Again, a large increase in c-Jun was observed after EGF treatment in KITENIN-transfected SW-620 cells than in co-vector transfected cells (Fig. 2D, top panel). Compared with Caco2 and HCT116 cells expressing EGFR, SW-620 cells showed very low chemotactic response to fibronectin; Anchorage-independent cell growth was increased after EGF treatment in KITENIN-transfected SW-620 cells (Fig. 2D, bottom panel). EGF is thought to induce increased c-Jun / AP-1 activity and cell migration in EGFR-independent manner in KITENIN-overexpressing CRC cells. Next, we examined whether the increase of AP-1 activity by EGF was induced from JNK activity under KITENIN transfection. In HCT116 cells (11) expressing the highest level of endogenous KITENIN among CRC cells, there was no substantial change in phospho-JNK despite the increase of phospho-c-Jun after EGF treatment (panel A of FIG. 9). In addition, JNK blockade through pretreatment of SP600125 with KITENIN transfected 293T cells had no effect on the increase of c-Jun by EGF (panel B of FIG. 9). Thus, EGF induced an increase in AP-1 activity / cell migration under KITENIN expression, which does not require EGFR-kinase or activated JNK.

EGF induces AP-I elevation through downregulation of KITENIN-coupled phospho-Dvl2 and upregulation of consecutive c-Jun.

Next, the underlying mechanism of AP-1 activation (AP-1 synergy) by EGF under KITENIN-transfection was investigated. EGFR downstream signaling pathways (5, 6), such as PI3K-mTOR and KRAS-RAF-MAPK pathways, were excluded from possible candidates since EGF induces AP-I synergy in an EGFR kinase- and EGFR-independent manner. Based on the hypothesis that PMA acts as an upstream signal for the functional KITENIN complex (where KITNEIN induces ERK / AP-1 activation through interaction with Dvls / PKCδ) (11) To affect Dvl on AP-1 activation. PMA induced a 5-fold increase in c-Jun, but at the same time decreased total Dvl2 by 70% compared to maternal cells in KITENIN-transfected 293T cells (Figure 3A), suggesting that c-Jun / Lt; RTI ID = 0.0 &gt; AP-1 &lt; / RTI &gt; To investigate the possibility that Dvl2 levels might contribute to increased c-Jun / AP-1 activity, Dvl2 levels were regulated in 293T cells by examining AP-1 / TOPflash reporter activity and c-Jun levels. Dvl2-knockdown increased AP-1 activity and c-Jun protein, while increased Dvl2 upregulated TOPflash, but did not upregulate AP-1 activity (panel A of FIG. 10). Next, we evaluated whether EGF could increase c-Jun in CRC cells as well as PMA. Since no phospho-Dvl2-specific antibody was available, HCT116 cells were selected and the changes in Dvl2 were analyzed under phosphatase treatment. Two hours after EGF treatment, the density of the phospho-Dvl2 and unphospho-Dvl2 levels was reduced to half (Figure 3B, top panel), while the level of c-Jun increased by three times as much as before treatment. EGF also showed a 3-fold increase in c-Jun and a 70% decrease in total Dvl2 in the KITENIN-transfected 293T cells (panel B in FIG. 10) as compared to the parent cells. In addition, after EGF treatment in KITENIN transfected Caco2 cells, c-Jun increased by 120% and total Dvl2 decreased by 80% (panel C of FIG. 10). In addition, a decrease in c-Jun and Dvl2 levels was observed after EGF treatment in KITENIN-transfected EGFR-deficient MEF and SW-620 cells compared to before treatment (Figs. 2C and 2D). To determine whether reduced Dvl2 and increased c-Jun were responsible for EGF or AP-1 synergy by PMA under KITENIN-transfection, Dvl2 or dominant negative c-Jun (DN-c-Jun ). &Lt; / RTI &gt;

Transfected Dvl2 or DN-c-Jun reversed upregulated AP-1 activity by EGF or PMA (Fig. 3C), suggesting that downregulation of Dvl2 and upregulation of c- Induced &lt; RTI ID = 0.0 &gt; AP-I &lt; / RTI &gt; synergy in overexpressed cells. Next, the nature of the interaction between KITENIN and Dvl2 after EGF treatment was investigated. Only Phospho-Dvl2 interacted with KITENIN; After the phosphatase treatment, the interaction between KITENIN and Dvl2 disappeared (FIG. 3B, bottom panel). At 30 minutes after EGF treatment in HCT116 cells, the level of KITENIN-bound phospho-Dvl2 initially increased but decreased after 2 hours. Consistently, at 30 minutes after EGF treatment in 293T and HCT116 cells, it was observed that KITENIN migrated from the cell membrane to the cytoplasmic ER containing phospho-Dvl2 (FIG. 3D, KITENIN panels). Importantly, treatment of EGF resulted in an increase in the number of co-existing endoplasmic reticulum (cytoplasmic dots), indicating that KITENIN associated with the membrane is internalized and subsequently interacts with phospho-Dvl2. Therefore, it was confirmed that the decrease of phospho-Dvl2 was caused by c-Jun increase rather than total Dvl2. When phosphorylation of Dvl was blocked by treatment with casein kinase I inhibitor D4476, phospho-c-Jun / total c-Jun was upregulated, whereas c-Fos was not (Fig. 3E, left panel). In contrast, co-transfection of casein kinase I not only attenuated AP-1 synergy by EGF under KITENIN-transfection or by PMA, but also after EGF treatment under KITENIN transfection or after PMA treatment Restored increased c-Jun and reduced total Dvl2 (Figure 3E, right panel). We interpreted the results as follows: EGF first induces endocytosis of KITENIN and then activates KITENIN / phospho-Dvl2 complex formation in cytoplasmic ER. EGF then down-regulates the KITENIN-coupled phospho-Dvl2 and then contributes to increased c-Jun / AP-1 activity. Interestingly, Dvl2-knockdown did not increase transcription of c-Jun, and Dvl2 did not interact directly with c-Jun (panel A and panel B of Figure 11). JNK blockade also did not affect EGF action in KITENIN transfected cells (panel B of FIG. 9). These results suggest that down-regulation of phospho-Dvl2 after EGF treatment leads to AP-1 synergy through stabilization of c-Jun protein. It is not currently known how the reduction of the KITENIN-bound phospho-Dvl2 stabilizes c-Jun, but this mechanism has been shown to be involved in N-terminal phosphorylation of c-Jun by JNK or phospho-c- It is distinct from the known regulator of Jun (21, 22).

ErbB4 is required for EGF-induced AP-1 synergy.

KITENIN can assist in the processing of KITENIN-coupled Dvl2, since EGFR is not essential for EGF-induced AP-I synergy and it has been confirmed that downregulation of KITENIN-linked phospho-Dvl2 is responsible for this EGF action (RTKs) as co-regulators of other receptor tyrosine kinases (RTKs). To investigate whether expression levels of KITENIN could affect the activation of RTKs, phosphorylation levels of various RTKs were compared between maternal cells and KITENIN-overexpressing CRC cells and between B cells and KITENIN-knockdown CRC cells. In KITENIN-overexpressed Caco2 cells, phospho-RTKs including ErbB4 and RON were increased, while phospho-Ephrin1 was decreased. On the other hand, phospho-ErbB4, phospho-RON, and phospho-c-Ret decreased in KITENIN knockdown HCT116 cells (panel A of FIG. 12). Phospho-c-Met was detected constantly in HCT116 cells. Thus, among the phospho-RTKs studied, RON and ErbB4 were consistently confirmed to be affected by KITENIN. As expected, the phospho-ErbB4 and phospho-RON forms, which are not in their total form, increased in Caco2 / KITENIN cells and decreased in HCT116 / si-KITENIN cells (panel B of FIG. 12). Interestingly, the increase in infiltration of Caco2 / KITENIN cells, indicating functional enhancement, as compared to the parent cells, was restored by ErbB4-knockdown but not by RON-knockdown (panel A of FIG. 4). These results indicate that ErbB4 is an essential cofactor in KITENIN signaling and increases CRC cell infiltration. We then investigated whether ErbB4 is required as a binding partner to aid in KITENIN signaling. KITENIN interacts with ErbB4 through the C-terminal cytoplasmic domain (panel C of FIG. 12), and that the interaction between KITENIN and ErbB4 is increased by EGF treatment (panel B of FIG. 4). Importantly, ErbB4-knockdown attenuated EGF-induced AP-1 synergy in KITENIN-transfected cells (Panel C in FIG. 4), suggesting that ErbB4 is required for EGF-induced AP-1 synergy . In addition, EGF treatment induced a significant increase in AP-1 activity in KITENIN / ErbB4-co-transfected cells compared to cells transfected with EGFR alone, ErbB4 alone, or KITENIN / EGFR 13). Consistently, ErbB4-knockdown reduced the interaction between KITENIN and Dvl2 (panel D, top panel of FIG. 4) and increased EGF-induced c-Jun, unaffected by EGFR- (Panel D, bottom panel of Figure 4). Thus, the present inventors have found that ErbB4 is essential for increasing the KITENIN-Dv12 interaction after EGF treatment and for facilitating the processing of KITENIN-coupled Dvl2, thereby resulting in the increase of c-Jun increase after EGF treatment under KITENIN- It is confirmed that it is the cause. In addition, EGF promoted the formation of the KITENIN / ErbB4 complex.

The EGF-KITENIN / ErbB4-c-Jun axis upregulates CRC cell infiltration.

Whether the EGF-KITENIN / ErbB4-c-Jun axis acts in an EGFR-independent / ErbB4-dependent manner was investigated using CHO (Chinese hamster ovary) cells, an ErbB-deleted cell line. In the KITENIN / ErbB4-cotransfected CHO cells than in the parental or KITENIN single transfected cells, we observed further increased c-Jun and Dvl2 reduction after EGF treatment (Fig. 5A). This further confirmed that the KITENIN / ErbB4-mediated downstream signal of EGF increases c-Jun in an EGFR-independent manner. In addition, it was shown that c-Jun increases after EGF treatment in both KITENIN-transfected EGFR-knockout cells (Fig. 2C) and EGFR-disappeared SW-620 cells (Fig. 2D), KITENIN / EGFR- (Figure 13) was observed between EGFR-only cells and EGFR-only-transfected cells after EGF treatment, indicating that the axis of EGF-KITENIN / ErbB4-c-Jun was asymmetric EGFR -ErbB4 heterodimer formation. &Lt; / RTI &gt;

Next, we investigated whether the ErbB4 / KITENIN co-expression could further enhance CRC cell infiltration as a result of enhancing the EGF-KITENIN / ErbB4-c-Jun axis. For this, HCT116 cells with the highest endogenous KITENIN levels and KRAS mutations were selected. Cellular infiltration was significantly greater in ErbB4-transfected HCT116 cells than in HCT116 / B cells when treated with EGF (Fig. 5B). However, this increased pattern disappeared after KITENIN-knockdown, although HCT116 cells still expressed increased ErbB4 expression. Thus, KITENIN may be a more important component than ErbB4 in controlling CRC cell invasion through the EGF-KITENIN / ErbB4-c-Jun axis. In addition, treatment with AG1478 in KITENIN / ErbB4-co-transfected Caco2 cells did not reduce the effect of EGF on cellular infiltration as in KITENIN-transfected Caco2 cells (Fig. 2B) 5C). These results confirm that upregulation of cellular invasiveness by EGF under cotransfected KITENIN / ErbB4 does not require the control of ErbB kinase activity.

KITENIN / ErbB4 is co-expressed in tumor tissue from metastatic CRC patients with poor response to cetuximab / chemotherapy.

Since 2004, only clinical anti-EGFR mAb has been used clinically to treat patients with metastatic CRC, but the response rate was less than 20% for KRAS wild-type patients (5-7). Therefore, we investigated the functional significance of the KITENIN / ErbB4 complex in this study in patients with cetuximab-resistant metastatic CRC. To this end, expression levels of KITENIN and ErbB4 were examined in resected tumor tissues from metastatic CRC patients. These patients exhibited wild-type KRAS and were treated primarily with cetuximab / chemotherapy (Table 2). Immunohistochemical coexpression of KITENIN / ErbB4 is more frequent in tumor tissues of metastatic CRC patients showing early stage (2 months) of disease development (PD) after treatment with cetuximab compared to patients with partial response (PR) Respectively. However, the expression level of ErbB4 in CRC patients with PR did not match that of KITENIN (panel A of FIG. 6). To quantitatively assess the immunohistochemical expression of KITENIN and ErbB4 in colon tissues, the scores were calculated using the intensity and size of the positively stained sites. KITENIN expression scores were significantly higher in tumor tissues from patients with PD than in patients with PR (8.05 +/- 0.30, n = 22 vs. 4.42 +/- 0.33, n = 33; P = 5.9) x 10 ^-10 ). ErbB4 was highly expressed in both groups of tumor tissues without significant difference in ErbB4 score (Panel B, Figure 6, 6.05 0.51, n = 22 vs. 6.18 0.41, n = 33; P = 0.84). Patients with good initial reactivity had a significantly longer progression free survival (PFS) than patients with poor response (Table 2, p = 0.014). Thus, the data of the present invention suggests that the initial chemical resistance to cetuximab in patients with high-KITENIN expression may be due to the presence of a unique EGF signal that is switched on under increased KITENIN conditions. The novel EGF signal described in this study may also provide enhanced survival rates in response to EGFR-targeted therapy to KITENIN high-expression CRC cells. To test this possibility, the sensitivity of various CRC cells to the first dose increase of cetuximab was examined. We have found that HCT116 and Caco2 cells expressing high levels of endogenous KITENIN are more resistant to cetuximab than DLD1 and SW620 cells expressing KITENIN at lower levels (panel A of FIG. 14). In order to test whether the high level of KITENIN expression affects the survival of CRC cells responding to cetuximab, Caco2 CRC cells, the KRAS / BRAF wild-type cell line (23), were first selected for use with EGFR targeting treatment (5) and (6), respectively. There was no difference in survival for cetuximab between co-vector and KITENIN-transfected Caco2 cells, whereas KITENIN-expressing Caco2 cells were resistant to cetuximab, whereas KITENIN knockdown Caco2 cells were sensitive (Panel B in Fig. 14). The proliferation rates for cetuximab were compared in CRC cells with mutant KRAS / BRAF such as DLD1 and HCT116 cells. KITENIN High expressing DLD1 cell line showed resistance to cetuximab, while KITENIN-knockdown HCT116 cells showed sensitivity to cetuximab (FIG. 14, panel C). Thus, these results indicate that the KITENIN / ErbB4-c-Jun axis confers resistance to cetuximab in Caco2 cells with KRAS / BRAF wild type and high KITENIN expression. These results also suggest that these axons may contribute to the initial acquisition of resistance to cetuximab in CRC cells with mutant KRAS / BRAF.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Industry Foundation of Chonnam National University <120> Screening Method for Therapeutic Agent Capable of Treating Cancer          Having Resistance to Epidermal Growth Factor Receptor Blocking          Agent <130> PN140402 <160> 16 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 1 ctacaatgta gatggcccc 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 2 agcctcatca ctgacaagcc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 3 tggaaaacct gcagatcatc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 4 ttgctgagaa agtcactgct 20 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 5 cagtgtgaga aaatggaaga tggactcctc 30 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 6 gcacgtttct ttttgatgct ctttcttctg 30 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 7 aagaatggct agagaccctc a 21 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 8 ggaccgctgg aagcactgtg ata 23 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 9 cgacggccac tgctctcaca t 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 10 atgacaactg gggagagacc a 21 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 11 cagctcctcc ctccacagga 20 <210> 12 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 12 gcuuggacuu cagccucgua gucaa 25 <210> 13 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 13 aacuuugaga acaugagcaa c 21 <210> 14 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 14 ugaucuguca ccacauaauu acggg 25 <210> 15 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 15 uauagauguu uccugcgcug auuuc 25 <210> 16 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 16 ugaaagagaa gccucucagc acgga 25

Claims (15)

A method for screening an anticancer agent candidate comprising the steps of:
(a) treatment of candidate substances with the treatment of epidermal growth factor (EGF) with cells having a signaling pathway of KITENIN / ErbB4-Dvl2-c-Jun axis, step;
(b) measuring the degree of activation of the signal transduction pathway of the KITENIN / ErbB4-Dvl2-c-Jun axis in the treated cells; And
(c) When activation of the signaling pathway of the KITENIN / ErbB4-Dvl2-c-Jun axis is further inhibited in the cells treated with the candidate substance compared to the cells not treated with the candidate substance, the candidate substance is selected as an anticancer drug candidate Judging step.
The method according to claim 1, wherein the cancer is cancer that is not responsive to an epidermal growth factor receptor (EGFR) blocking agent.
The method of claim 1, wherein the cancer is gastric cancer, liver cancer, or colon cancer.
The method according to claim 1, wherein the inhibition of the activation of the signaling pathway of the KITENIN / ErbB4-Dvl2-c-Jun axis in step (c) comprises (i) inhibition of binding of KITENIN to ErbB4, (ii) (Iii) inhibition of an increase in the amount of c-Jun protein, or (iv) inhibition of AP-1 transcription factor activation.
Epithelial growth factor (EGF); And KITENIN / ErbB4-Dvl2-c-Jun axis.
6. The composition of claim 5, wherein the cancer is a cancer that is not responsive to an epithelial growth factor receptor (EGFR) blocking agent.
6. The composition of claim 5, wherein the cancer is gastric cancer, liver cancer or colon cancer.
delete delete delete (EGFR) blocking agent, which comprises measuring the degree of activation of the signaling pathway of the KITENIN / ErbB4-Dvl2-c-Jun axis in cancer tissues or cells, or determining the reactivity to an EGFR blocking agent How to provide information necessary for prognosis.
12. The method of claim 11, wherein measuring the degree of activation of the signaling pathway of the KITENIN / ErbB4-Dvl2-c-Jun axis comprises measuring the amount of KITENIN protein.
12. The method of claim 11, wherein the cancer is gastric cancer, liver cancer, or colon cancer.
KITENIN kit for determining the reactivity of a cancer containing a protein detection reagent to an epithelial cell growth factor receptor (EGFR) blocking agent.
15. The kit according to claim 14, wherein the cancer is gastric cancer, liver cancer or colon cancer.

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