WO2022255401A1 - Disease marker expressed in association with abnormal erk-mapk pathway activation - Google Patents

Abstract

The present invention provides disease markers, in particular, a cancer detection marker and a Ras/MAPK syndrome detection marker. Specifically, the present invention is a cancer detection marker containing at least one type of molecule selected from a protein group, the expression of which is induced by ERK1 or ERK2, the cancer detection marker being characterized in that the ERK1 and the ERK2 are activated by an MEK1 mutant or an MEK2 mutant activated by self-phosphorylation of a T-loop region. In addition, the present invention is a Ras/MAPK syndrome detection marker containing at least one type of molecule selected from a protein group, the expression of which is induced by ERK1 or ERK2, the Ras/MAPK syndrome detection marker being characterized in that the ERK1 and the ERK2 are activated by an MEK1 mutant or an MEK2 mutant exhibiting activities even if a T-loop region is not phosphorylated.

Description

Disease marker expressed with abnormal activation of ERK-MAPK pathway

The present invention relates to disease markers.

Various cancer marker molecules have been reported so far for the purpose of cancer diagnosis and post-treatment follow-up. Some of them have already been put to practical use, for example, blood PSA (prostate specific antigen) is used as a marker molecule for prostate cancer. However, the expression levels of many other cancer marker molecules do not necessarily match the development of cancer. In particular, there are no effective diagnostic markers yet for pancreatic cancer, and the difficulty in early detection is a cause of poor prognosis.
As a cause of such various cancers, many reports have been reported so far on activating mutations/gene amplifications of Ras, Raf, MEK, etc. that constitute the ERK-MAP kinase pathway (Raf-MEK-ERK) (pancreatic cancer). , malignant melanoma, colon cancer, thyroid cancer, lung cancer, ovarian cancer, etc.). In fact, KRas is mutated to an active form in more than 90% of pancreatic cancers, and BRaf is mutated to an active form in about 50% of metastatic melanomas, constitutively activating the ERK pathway. In addition, in BRaf-activated malignant melanoma and ALK-activated lung cancer, although molecular-targeted drugs temporarily disappear, the ERK pathway is eventually reactivated via collateral pathways. Acquisition of drug resistance has been shown to cause cancer recurrence. In addition, several missense mutations in MEK, one of the genes that make up the ERK-MAP kinase pathway, were identified by genetic analysis of lung cancer, colon cancer, ovarian cancer, and malignant melanoma (Non-Patent Document 1 and Non-Patent Document 2). In fact, the MEK1 (K57N) mutation found in lung tumors has been shown to significantly promote cell proliferation through activation of the ERK pathway (Non-Patent Document 3).

So far, it has been clarified that genetic mutations in the constituent molecules of the ERK-MAP kinase pathway aberrantly activate the pathway, leading to carcinogenesis. Abnormal activation of the ERK-MAP kinase pathway is also involved in the acquisition of drug resistance and recurrence of cancer cells. Furthermore, in recent years, activating mutations such as Raf and MEK in the germ line have been identified as the cause of congenital Ras/MAPK syndrome, which can lead to multiple abnormalities such as heart failure, skin and skeletal muscle abnormalities, and mental retardation. reported (Non-Patent Document 4).
Therefore, if a molecular marker that can sensitively detect abnormal activation of the ERK-MAP kinase pathway can be found, it can be used for early diagnosis of cancer and Ras/MAPK syndrome and prediction of cancer recurrence. However, at present, it is not clear whether any abnormalities in the ERK-MAP kinase pathway are related to cancer or Ras/MAPK syndrome, and it is one of the important problems to be solved in the medical field. be.

In view of the above circumstances, the present invention clarifies what diseases are triggered by abnormal activation of the ERK-MAPK pathway and what gene expression is induced. It is an object of the present invention to provide markers for early detection of activation-related diseases, particularly cancer, and early detection of Ras/MAPK syndrome.

We show that identified MEK mutations in the MEK1/2 genes from cancer and the Ras/MAPK syndrome enhance the kinase activity of MEK by affecting phosphorylation, a post-translational modification I found out. Specifically, the present inventors demonstrated that cancer-derived MEK mutants exhibit constitutive and strong kinase activity due to intramolecular autophosphorylation of the T-loop, and that the Ras/MAPK syndrome-derived MEK We found for the first time that mutants have constitutive kinase activity even in the unphosphorylated state of the T-loop.
Based on the above findings, the inventors attempted to search for genes that are highly expressed only when the ERK pathway is constitutively and strongly activated, among genes whose expression is induced downstream of the ERK pathway. . Human-derived epithelial cells were transfected with cancer-derived active MEK1 point mutants or Ras/MAPK syndrome-derived active MEK1 point mutants, and cDNA microarray analysis was performed to obtain genome-wide gene expression profiles in the cells. analyzed exhaustively. As a result, we found that the expression levels of various mRNAs fluctuate greatly.
In particular, regarding cancer-derived MEK1 mutants, we collected various cancer cells (malignant melanoma, lung cancer, pancreatic cancer, colorectal cancer, etc.) in which abnormal activation of the ERK pathway was observed, and examined these genes. When we monitored the expression level at the RNA and protein levels, we confirmed that it was actually strongly expressed in most cancer cell lines. In addition, for some molecules, analysis (immunostaining) using clinical cancer tissue is also performed, and at least in clinical samples of pancreatic cancer, lung cancer, and colon cancer, at high frequency (in 80% or more cases) High expression was confirmed. Interestingly, many of these molecules included secretory proteins and membrane proteins that could serve as novel cancer detection markers. These proteins contribute to the diagnosis and early detection of various sporadic cancers (malignant melanoma, colorectal cancer, thyroid cancer, lung cancer, ovarian cancer, etc.) caused by abnormal activation of the ERK pathway. It is thought that it is possible to utilize it as a diagnostic marker molecule.
The present invention has been completed based on the above findings.

That is, the present invention is the following (1) to (13).
(1) A cancer detection marker comprising at least one molecule selected from a group of proteins whose expression is induced by ERK1 or ERK2, wherein said ERK1 and ERK2 are activated by autophosphorylation of the T-loop region. A marker for cancer detection characterized by being activated by a MEK1 mutant or MEK2 mutant.
(2) The marker for cancer detection according to (1) above, wherein the mutation in the MEK1 mutant is Q56P, K57N, C121S or E203K.
(3) The marker for cancer detection according to (1) above, wherein the mutation in the MEK2 mutant is Q60P.
(4) MMP10, EMP1, Rheb2, TM4SF1, TM4SF19, TMEM158, ENDOD1, c2orf89, SLC20A1, LY6K, PLAUR (CD87), PVR (CD155), IL7R (CD127), IL1R2 (CD121b), IL4R, TweakR (CD266), CD3D, CD44, SEMA7, IL13RA2, THBD, XAGE1, PRR9, TRIB1, IER3, c11orf96, c8orf4, PHLDA1, PHLDA2, DUSP5, DUSP6, ERRFI1, GADD45B, IER3, IRX4, SPANXN3, SPANXN4, SPANXN5, TGFb1, BMP2, TFPI2, The cancer detection marker according to (1) above, comprising at least one protein selected from the group consisting of GDF15, PAEP, CCL7, IL11 and CRLF.
(5) The cancer detection marker according to (4) above, comprising at least one protein selected from the group consisting of EMP1, TM4SF1, TM4SF19, c11orf96, PHLDA1, PHLDA2, TFPI2, Rheb2 and GDF15.
(6) MMP10, EMP1, Rheb2, TM4SF1, TM4SF19, TMEM158, ENDOD1, c2orf89, SLC20A1, LY6K, PLAUR (CD87), PVR (CD155), IL7R (CD127), IL1R2 (CD121b), IL4R, TweakR (CD266), CD3D, CD44, SEMA7, IL13RA2, THBD, XAGE1, PRR9, TRIB1, IER3, c11orf96, c8orf4, PHLDA1, PHLDA2, DUSP5, DUSP6, ERRFI1, GADD45B, IER3, IRX4, SPANXN3, SPANXN4, SPANXN5, TGFb1, BMP2, TFPI2, A cancer detection kit comprising antibodies or aptamers against GDF15, PAEP, CCL7, IL11 or CRLF.
(7) A method for diagnosing cancer or diagnosing cancer, comprising the step of measuring the expression level of the cancer detection marker according to any one of (1) to (6) above present in a sample derived from a subject. Auxiliary method.
(8) A marker for cancer detection, comprising the step of searching for a protein that induces the expression of ERK1 or ERK2 that is activated by a MEK1 mutant or MEK2 mutant that is activated by autophosphorylation of the T-loop region. screening method.
(9) A marker for detecting Ras/MAPK syndrome containing at least one molecule selected from the group of proteins whose expression is induced by ERK1 or ERK2, wherein the ERK1 and ERK2 are not phosphorylated in the T-loop region A marker for detecting Ras/MAPK syndrome, characterized by being activated by a MEK1 mutant or MEK2 mutant having activity even in the case of Ras/MAPK syndrome.
(10) The above ( 9) Ras/MAPK syndrome detection marker.
(11) The mutation in the MEK2 mutant is F53C, F53V, F57L, K61E, A62P, P128R, G132V, T134C, or Y134H. For detecting Ras / MAPK syndrome according to (9) above. marker.
(12) comprising the step of measuring the expression level of the Ras/MAPK syndrome detection marker according to any one of (9) to (11) present in a subject-derived sample, a method for diagnosing Ras/MAPK syndrome or A diagnostic aid for Ras/MAPK syndrome.
(13) Ras/MAPK, including the step of searching for proteins that induce the expression of ERK1 or ERK2 activated by MEK1 or MEK2 mutants that are active even if the T-loop region is not phosphorylated Screening method for markers for syndrome detection.
In this specification, the sign "-" indicates a numerical range including the values on the left and right of it.

By using the novel cancer detection marker according to the present invention, various cancer types that could not be accurately diagnosed with existing markers, especially cancers caused by abnormal activation of the ERK pathway (pancreatic cancer, malignant melanoma, colon cancer, thyroid cancer, lung cancer, breast cancer, stomach cancer, ovarian cancer, sarcoma, etc.) can be detected with high sensitivity and accuracy.

In addition, early diagnosis and detection of cancer is possible using the novel cancer detection marker according to the present invention.

Furthermore, by using the Ras/MAPK syndrome detection marker according to the present invention, early diagnosis and detection of Ras/MAPK syndrome, which is difficult to diagnose, becomes possible.

Kinase activity of MEK mutants. (A) shows the results of measuring the kinase activity (using ERK2 as a substrate) of cancer-derived MEK1 mutants and other MEK1 mutants. Kinase-deficient Myc-ERK2(K/N) and HA-MEK1 were co-transfected into HEK293 cells. Phosphorylated ERK2 was detected by immunoblotting and its band intensity was quantified (P-ERK). The same filter was blotted again with an anti-Myc antibody to confirm the amount of ERK present. Mutants with Q56P and K57N mutations are cancer-derived MEK1 mutants. Other variants are those derived from the Ras/MAPK syndrome. (B and C) Blotting with anti-phospho-MEK antibody (P-MEK) against HA-MEK1 mutant transfected into HEK293 cells (B) or recombinant GST-MEK1 mutant purified from E. coli (C). did (D) Results showing that Ras/MAPK syndrome-derived MEK mutations are activated independently of phosphorylation. Affinity-purified GST-MEK1 or its mutant proteins were incubated in the presence or absence of BRaf(V600E) protein, followed by incubation with GST-ERK2(K/N). K/M; K97M mutation that lacks kinase activity; AA; non-phosphorylated mutant in which Ser218 and Ser222 of the T-loop are replaced with alanine. Autophosphorylation of the T-loop region of MEK1 mutants. (A) GST-MEK1 or cancer-derived MEK1 mutants (Q56P or K57N) were purified from E. coli and immunoblotted with an anti-phospho-MEK antibody. K/M; indicates the kinase activity-deficient K97M mutation. (B) HA-MEK1 or its mutants (Q56P and K57N) were transiently expressed in HEK293 cells, and T-loop phosphorylation was detected in the same manner as in (A). (C) Purified MEK1 protein was incubated with ERK2 (K/N) lacking kinase activity, and ERK phosphorylation was detected by blotting with anti-P-ERK antibody. The band intensity of P-ERK was quantified. (D) GST-MEK1(Q56P) mutants were incubated with kinase activity-deficient MEK1(K/M) or MEK2(K/M), phosphorylation of MEK (GST-MEK1(Q56P)) was inhibited by anti-P-MEK detected with an antibody. AA represents non-phosphorylated mutations. (E) Myc-MEK1(Q56P) was transiently expressed in HEK293 cells with kinase activity deficient HA-MEK1(K/M) or HA-MEK2(K/M). TPA-treated HEK293 cells (300 nM, 20 min treatment) served as a positive control for MEK phosphorylation. Autophosphorylation of the T-loop region of MEK2 mutants. (A) GST-MEK2 or one of the mutants (MEK2(Q60P) and MEK2(K61E)) was expressed in E. coli and its T-loop phosphorylation was detected by blotting. K/M represents mutations that lack kinase activity. A62P is a RAS/MAPK syndrome-derived variant. (B) HEK293 cells were transfected with either HA-MEK2 or its mutants, and T-loop phosphorylation was detected with a P-MEK antibody. K/M represents mutations that lack kinase activity. Effects of MEK mutations on ERK pathways. (A) HEK293 cells expressing HA-MEK1(WT), HA-MEK1(F53S) (derived from Ras/MAPK syndrome) or HA-MEK1(K57N) (derived from cancer) were stimulated with EGF (5 ng/ml). did. Phosphorylation of ERK (“P-ERK”), MEK (“P-MEK”), Raf-1 (“P-Raf1(S338)”) and S6 ribosomal protein (“P-S6K”) after stimulation with EGF was detected with a phosphorylated antibody. Egr1 expression levels were also monitored (“Egr1”). (B) HEK293 cell lines stably co-expressing ERK1-GFP and various MEK1 proteins were stimulated with EGF (5 ng/ml), and ERK localization was continuously monitored using a time-lapse imaging system. . The amount of ERK1-GFP fluorescence was quantified, and the percentage of ERK1 translocated to the nucleus was graphed. Error bars indicate SEM, n=5. (C) The expression level of Egr1 in (A) was graphed. Gene expression induced by MEK1 mutants from Ras/MAPK syndrome. (A) Relative expression levels of COL14A1 in HEK293 cells expressing MEK1 (WT) or its mutants (F53S or K57N) were quantified by q-PCR. (B) The Ras/MAPK syndrome-derived MEK1 mutant (F53S) had constitutive kinase activity and selectively induced the expression of collagen type 14 (COL14A1). This is the result of filtering the cell culture supernatant, separating with SDS-PAGE, and performing immunoblotting. Gene expression induced by cancer-derived MEK1 mutant (MEK1(K57N)). The results of quantitative PCR using total RNA samples derived from HEK293 cells stably expressing MEK (WT) or MEK mutants (F53S and K57N) are shown. Error bars indicate SEM (n=3). **, P < 0.01; *, P < 0.05. Confirmation of expression in cancer cells of gene products induced by cancer-derived MEK1 mutation (K57N). HEK293 (with MEK1(WT) or MEK1(K57N)), H1299 (NRas ^Q61K ) (from lung cancer), A375 (BRaf ^V600E ) (from malignant melanoma), sk-Mel28 (BRaf ^V600E ) and WiDr (BRaf ^V600E ) (Colon cancer-derived) cells were treated in the presence or absence of U0126 (20 μM, 24 h), and the results of blotting of cell lysates or cell culture supernatants with antibodies against each protein are shown. Expression levels of C11orf96 protein in human malignancies. Normal and malignant tissues of pancreas (left), lung (middle) and colon (right) were stained with anti-C11orf96 antibody (top). The brown staining intensity of c11orf96 in each tissue was classified into the following categories; negative (no staining or <10% weakly stained cells), weak (<10% strongly stained cells or weakly stained more than 10% of cells stained), strong (more than 10% of strongly stained cells). This classification is shown in a bar graph (right panel). Expression differences between normal and malignant tumor tissues were evaluated with a chi-square test. ***, P < 0.001; **, P < 0.01; *, P < 0.05. All pictures are shown at x400 magnification.

A first embodiment is a cancer detection marker comprising at least one molecule selected from a group of proteins whose expression is induced by ERK1 or ERK2, wherein the ERK1 and ERK2 autophosphorylate the T-loop region. A marker for cancer detection characterized by being activated by a MEK1 mutant or a MEK2 mutant that is activated by .
ERK1 and ERK2 are members of the MAPK (Mitogen-activated protein kinase) family, have molecular weights of 44 kDa and 42 kDa, respectively, and have approximately 85% homology in their amino acid sequences. In normal cells, Ras activated by extracellular growth factor stimulation binds to Raf and promotes its activation, and Raf phosphorylates and activates MEK, which then phosphorylates ERK. oxidize and activate. Activated ERK translocates from the cytoplasm to the nucleus and activates transcription factors such as ELK, CREB, c-Myc, c-Fos and Sp-1 to induce gene expression.

The inventors have found that cancer (cell)-derived MEK1 and MEK2 mutants have their T-loop regions phosphorylated by intramolecular autophosphorylation and exhibit constitutive and strong kinase activity (see below). MEK1 mutants and/or MEK2 mutants having such characteristics are also described as "MEK1/2 mutants according to the first embodiment") for the first time. Furthermore, the inventors found that ERK1 and ERK2 activated by this MEK1 mutant or MEK2 mutant induce the expression of various cancer cell-specific proteins through the activation of various transcription factors. I found In other words, cancer-derived MEK1/2 mutants, which are characterized by their activation by intramolecular autophosphorylation of the T-loop region, indirectly induce the expression of cancer cell-specific proteins. , the protein whose expression is induced can be used as an effective marker for detecting the presence of cancer cells (hereinafter also referred to as a "cancer detection marker according to the first embodiment"). .
In this specification, when MEK1/2 and ERK1/2 are described, they mainly refer to MEK1 protein and/or MEK2 protein, ERK1 protein and/or ERK2 protein. Make a note to that effect.

Here, the MEK1/2 T-loop region exists, for example, in the 208th to 233rd amino acid region on the MEK1 amino acid sequence and in the 212th to 237th amino acid region on the MEK2 amino acid sequence. In the MEK1 mutant and MEK2 mutant according to the first embodiment of the present invention, amino acid residues present in the T-loop region (for example, in the case of MEK1, Ser218, Ser222, etc.) are intramolecularly autophosphorylated. It is characterized by being activated, showing constant and strong kinase activity. MEK1/2 mutants according to the first embodiment include, but are not limited to, MEK1 mutants with Q56P, K57N, C121S or E203K mutations, and MEK2 mutants with Q60P mutations. can.

The MEK1/2 mutant according to the first embodiment may be naturally occurring or newly produced by genetic engineering or the like. Whether or not the MEK1/2 mutant has the ability to intramolecularly autophosphorylate can be determined, for example, by performing a kinase assay in which the MEK1/2 mutant and the MEK1/2 mutant that has lost the kinase activity are allowed to coexist. In this case, if the MEK1/2 mutant that has lost the kinase activity is not phosphorylated, it can be determined that the MEK1/2 mutant has intramolecular autophosphorylation ability (for details, see Examples checking).

A cancer detection marker according to the first embodiment or a cancer detection marker candidate molecule can be searched for, for example, as follows.
The MEK1/2 mutant according to the first embodiment is introduced into normal cells that are not cancerous, and the expression levels of various mRNAs of genes in the cells are comprehensively examined using, for example, microarrays and RNAseq. Analyzes are performed, and genes whose expression levels are statistically significantly increased are selected as compared with the expression levels in stably expressing cells transfected with wild-type MEK1/2. If the gene product (protein) of the selected gene is expressed in cancer cells or if the gene product expressed in cancer cells is present in serum-secreted exosomes, the molecule is It can be used as a marker for cancer detection.
Alternatively, when the cancer detection marker according to the first embodiment or the candidate molecule for the cancer detection marker is a protein, normal cells expressing the MEK1/2 mutant according to the first embodiment and , Proteome analysis (or quantitative proteome analysis) was performed on a sample prepared from normal cells expressing wild-type MEK1/2, and significantly expressed in normal cells expressing the MEK1/2 mutant according to the present embodiment. A protein is selected, and if the protein is expressed in cancer cells, that molecule can be used as a marker for cancer detection.

As a cancer detection marker according to the first embodiment, in cells expressing a cancer cell-derived MEK1 mutant (K57N) (one of the MEK1 mutants according to the first embodiment), The following proteins whose expression level is significantly increased can be exemplified.
membrane protein
EMP1, Rheb2, TM4SF1, TM4SF19, TMEM158, ENDOD1, c2orf89, SLC20A1, LY6K, PLAUR(CD87), PVR(CD155), IL7R(CD127), IL1R2(CD121b), IL4R, TweakR(CD266), CD3D, CD44, SEMA7 , IL13RA2, THBD
Cytoplasmic/nuclear proteins
XAGE1, PRR9, TRIB1, IER3, c11orf96, c8orf4, PHLDA1, PHLDA2, DUSP5, DUSP6, ERRFI1, GADD45B, IER3, IRX4, SPANXN3, SPANXN4, SPANXN5
secretory protein
MMP1, MMP10, TGFb1, BMP2, TFPI2, GDF15, PAEP, CCL7, IL11, CRLF

A second embodiment includes the step of searching for a protein that induces the expression of ERK1 or ERK2 that is activated by a MEK1 mutant or MEK2 mutant that is activated by autophosphorylation of the T-loop region. It is a screening method for cancer detection markers.
The search for molecules induced by MEK1 mutants activated by autophosphorylation of the T-loop region or ERK1 or ERK2 activated by MEK2 mutants can be performed by microarray analysis or proteome analysis, as described above. By the method, the expression levels of proteins expressed in cells introduced with the MEK1/2 mutant according to the first embodiment and cells introduced with the wild-type MEK1/2 are compared, and MEK1/2 according to the first embodiment It can be carried out by selecting a protein whose expression is significantly increased in cells into which two mutants have been introduced. Here, among the selected proteins, those that have been confirmed to be specifically expressed in cancer cells can be used as cancer detection markers.

A third embodiment provides a method for diagnosing cancer or diagnosing cancer, comprising the step of measuring the expression level of the cancer detection marker according to the first embodiment of the present invention present in a sample derived from a subject. It is an auxiliary method.
The cancer detection marker according to the first embodiment is not limited to specific cancers, and can be used for early detection of various cancers. For example, malignant melanoma, colon cancer, thyroid cancer, lung cancer, ovarian cancer, pancreatic cancer, breast cancer, gastric cancer, prostate cancer, bladder cancer, It can be used for early detection of blood cancer, sarcoma, and the like. In the method for diagnosing cancer and the method for assisting cancer diagnosis according to the third embodiment of the present invention, the abundance of the cancer detection marker according to the first embodiment in a sample derived from a subject is When the amount is statistically significantly higher than the abundance of the subject, it can be determined that the subject has or is likely to have some cancer. Examples of samples derived from a subject include tissues suspected of developing cancer, blood (including exosomes), and body fluids such as pancreatic fluid and urine. In addition, as a control sample, if the sample to be tested is a tissue, tissue or cells derived from the same tissue that have been confirmed to have the ERK pathway functioning normally, or cancer that has not been confirmed. Examples include body fluids such as blood, pancreatic fluid and urine derived from healthy subjects.

The fourth embodiment is a kit for cancer detection or cancer diagnosis. The kit of the present invention contains at least an antibody or aptamer for detecting the cancer detection marker according to the first embodiment. More specifically, for example, MMP10, EMP1, Rheb2, TM4SF19, TM4SF1, TMEM158, ENDOD1, c2orf89, SLC20A1, LY6K, PLAUR(CD87), PVR(CD155), IL7R(CD127), IL1R2(CD121b), IL4R, TweakR (CD266), CD3D, CD44, SEMA7, IL13RA2, THBD, XAGE1, PRR9, TRIB1, IER3, c11orf96, c8orf4, PHLDA1, PHLDA2, DUSP5, DUSP6, ERRFI1, GADD45B, IER3, IRX4, SPANXN3, SPANXN4, SPANXN5, TGFb1 , BMP2, TFPI2, GDF15, PAEP, CCL7, IL11 or CRLF may be included.

A fifth embodiment is a marker for detecting Ras/MAPK syndrome containing at least one molecule selected from the group consisting of proteins whose expression is induced by ERK1 or ERK2, wherein the ERK1 and ERK2 are T-loop regions A marker for detecting Ras/MAPK syndrome characterized by being activated by a MEK1 mutant or MEK2 mutant that has activity even if is not phosphorylated.
We found that MEK1 and MEK2 mutants from Ras/MAPK syndrome (germline cells derived from Ras/MAPK syndrome) constitutively have kinase activity even though their T-loop regions are not phosphorylated. (hereinafter, MEK1 and/or MEK2 mutants having such characteristics are also described as "MEK1/2 mutants according to the fifth embodiment"). Furthermore, the inventors found that ERK1/2 activated by this MEK1/2 mutant induces the expression of proteins specific to Ras/MAPK syndrome through the activation of various transcription factors. rice field. MEK1/2 mutants, which are characterized by having constitutive kinase activity even when the T-loop region is not phosphorylated, indirectly induce the expression of proteins specific to Ras/MAPK syndrome, The protein whose expression is induced is a marker effective for detecting the presence or absence or possibility of the onset of Ras/MAPK syndrome (hereinafter referred to as "marker for detecting Ras/MAPK syndrome according to the fifth embodiment of the present invention"). It is possible to use it as a
MEK1/2 mutants according to the fifth embodiment include, but are not limited to, MEK1 mutations having F53S, T55P, D67N, P124L, P124Q, G128V, G128N, Y130C, Y130N, Y130H, or E203Q mutations. MEK2 mutants with F53C, F53V, F57L, K61E, A62P, P128R, G132V, T134C, or Y134H mutations.

The MEK1/2 mutant according to the fifth embodiment may be naturally occurring or newly produced by genetic manipulation or the like.
As a marker for detecting Ras / MAPK syndrome according to the fifth embodiment, in cells expressing Ras / MAPK syndrome-derived MEK1 mutant (F53S) (one of the MEK1 mutants according to the fifth embodiment) , the Col14A1 protein whose expression level was significantly increased can be exemplified.

A sixth embodiment includes the step of searching for a protein that induces the expression of ERK1 or ERK2 activated by a MEK1 mutant or MEK2 mutant that has activity even if the T-loop region is not phosphorylated. , a screening method for markers for detecting Ras/MAPK syndrome.
Molecules induced by ERK1 or ERK2, which is activated by MEK1 or MEK2 mutants that are active even if the T-loop region is not phosphorylated, can be explored by methods such as microarray analysis or proteome analysis. The expression levels of proteins expressed in cells introduced with the MEK1/2 mutant according to the fifth embodiment and in cells introduced with the wild-type MEK1/2 are compared, and the MEK1/2 mutant according to the fifth embodiment can be carried out by selecting a protein whose expression is significantly increased in cells into which is introduced.

A seventh embodiment comprises the step of measuring the expression level of a Ras/MAPK syndrome detection marker according to the fifth embodiment of the present invention present in a sample derived from a subject, a method for diagnosing Ras/MAPK syndrome or It is a diagnostic aid for Ras/MAPK syndrome.
Here, Ras/MAPK syndrome is characterized by specific facial features (interocular dissociation, oblique palpebral fissure, temporal stenosis, etc.), cardiac hypertrophy, mental retardation, and cutaneous symptoms. It is a chromosomal dominant genetic disease (generic term for Costello/Noonan/CFC syndrome, etc.) (Hum Mutat. 2008 Aug;29(8):992-1006).
In the method for diagnosing Ras/MAPK syndrome and the method for assisting diagnosis of Ras/MAPK syndrome according to the seventh embodiment, the amount of the marker for detecting Ras/MAPK syndrome according to the fifth embodiment in a sample derived from a subject is A determination is made that the subject has or is at risk of having some form of Ras/MAPK syndrome if it is statistically significantly greater than the abundance in the control sample. be able to. Here, as a subject-derived sample, for example, by targeting amniotic fluid collected from a pregnant subject, there is a possibility that a newborn can be diagnosed congenitally with Ras/MAPK syndrome before birth. .

When the cancer detection marker according to the first embodiment and the Ras/MAPK syndrome detection marker according to the fifth embodiment are proteins, their expression levels can be detected and measured by known methods. Examples of such methods include methods using antibodies or aptamers (such as nucleic acid aptamers or peptide aptamers) that specifically bind to the cancer detection marker protein or Ras/MAPK syndrome detection marker protein used. More specifically, the detection and quantification of marker proteins can be performed by immunological techniques or methods utilizing the binding properties of antibodies or aptamers to marker proteins, such as immunochromatography and Western blotting. , ELISA (e.g. direct competitive ELISA, indirect competitive ELISA, sandwich ELISA, etc.), radioimmunoassay (RIA), fluorescence immunoassay (FIA), immunohistochemical staining, quartz crystal microbalance (Quartz It can be easily carried out by methods well known to those skilled in the art, such as methods using crystal microbalance (QCM) and magnetic beads.

Where this specification has been translated into English and contains the words "a", "an" and "the" in the singular, unless the context clearly indicates otherwise, the singular as well as the singular It also includes multiple items.
EXAMPLES The present invention will be further described below with reference to Examples, but these Examples are merely illustrations of embodiments of the present invention and do not limit the scope of the present invention.

1. Materials and experimental methods
In vitro kinase assay Purified MEK1/2 or various MEK1/2 mutants (0.6 μg) were mixed with GST-ERK2(K/N) (an ERK2 mutant that lost kinase activity) (0.6 μg) and the mixture was added to 160 μM ATP. Incubated at 26°C for 7 minutes in a kinase buffer containing The phosphorylation levels of T185 and Y187 of ERK2 were quantified using a luminescent image analyzer, LAS-1000 Plus (Fujifilm).

Observation of nuclear translocation of ERK HEK293 cells stably expressing ERK1-GFP and either HA-MEK1 (WT), HA-MEK1 (F53S) or HA-MEK1 (K57N) were seeded in glass bottom dishes and C. overnight to allow cells to adhere. ERK1-GFP fluorescence from cells in randomly selected wells was monitored every 90 seconds. Specifically, EGF was added to the medium to a final concentration of 5 ng/ml, and fluorescence from living cells was observed continuously for 60 minutes using a Nikon Eclipse Ti fluorescence microscope (Rolera EM- ^C2) equipped with a CCD camera. , QImaging). The ratio of ERK-GFP translocated to the nucleus in each cell was calculated by dividing the fluorescence intensity of the whole cell by the fluorescence intensity of the nuclear region of the same cell. Quantitative analysis of fluorescence intensity was performed using MetaFluor software (Molecular Devices).

Immunohistochemistry The three cancer tissue microarrays used in this example (pancreas; PA484, lung; LC483 and colon; CO483) were purchased from Biomax US. Tissue slides were deparaffinized and antigen-retrieved by incubation in citrate buffer (pH 6) at 95°C for 40 minutes. Endogenous peroxidase activity in each tissue sample was blocked by treatment with 0.3% hydrogen peroxide. Rehydrated tissue slides were incubated with BlockAce (Yukijirushi) for 10 minutes at room temperature, followed by anti-C11orf96 antibody (1:250 dilution) overnight at 4°C. An anti-rabbit-HRP antibody (Dako Envision systems) was used as a secondary antibody, and DAB (3,3'-Diaminobenzidine) was used to visualize the binding between the primary antibody and the antigen. Immunohistological evaluation of C11orf96 was performed by a pathologist.

Plasmid pcDNA3HA, pcDNA3Flag and pcDNA4Myc vectors were used to express MEK1, MEK2, ERK2, BRaf, Raf-1, KSR1 and their mutants. MEK1/2 mutants were generated by site-directed mutagenesis by PCR.
pCold (Takara Bio, Japan), pRSF-Duet (Merck-Millipore) and pGEX-6P vectors (GE healthcare) are used to express GST or His-tagged B-Raf, MEK1, MEK2 and ERK2 proteins in E. coli did. The catalytically inactive form of ERK2 (K/N) replaces the codon of Lys52 with that of asparagine, and the MEK1 (K/M) and MEK2 (K/M) inactive mutants replace codons of Lys97 and Lys101, respectively. was prepared by substituting the codon for methionine. Raf-1ΔN was produced as previously reported (Takekawa et al., Mol Cell 18, 295-306 2005).
For use of the Shield1-dependent protein stabilization system, the DD sequence (destabilization region, Clontech) was fused to the N-terminal side of the active MEK1 mutant (S218D/S222D), and the fused construct was cloned into the pQCXIP vector.

Media and Buffers In vitro kinase assays used a buffer containing 25 mM Tris-HCl (pH 7.5), 25 mM _MgCl2 , 10 mM β-glycerophosphate, 0.5 mM sodium vanadate and 2 mM DTT.

Tissue culture and transiently transfected immortalized MEK1-/- MEFs were kindly provided by J. Charron (Universite Laval, Quebec) (Giroux et al., 1999). Cells were maintained in high glucose DMEM containing 10% fetal bovine serum (FBS), L-glutamine, penicillin and streptomycin. HEK293 cells were cultured in low glucose DMEM containing 10% fetal bovine serum (FBS), L-glutamine, penicillin and streptomycin.
For transient transfections, HEK293 cells cultured in 35 mm dishes were transfected with expression plasmids using X-tremeGENE 9 DNA transfection reagent (Sigma). The total amount of DNA was 1 μg/dish. Cells were harvested 48 hours after transfection.

Retrovirus infection HEK293 cell lines stably expressing MEK mutants were generated by retrovirus infection. Retroviruses were generated in GP2-293 packaging cells by transient transfection with pVSV and pQCXIP or pQCXIH plasmids. Culture supernatants were harvested 48 hours after transfection, filtered and added with 8 μg/ml polybrene. Cells were retrovirally infected and cells expressing the protein of interest were selected with puromycin or hygromycin. Retroviral infection of MEFs was performed as described above, except Plat-E packaging cells were used.

Expression and Purification of Recombinant Proteins GST-MEK1, GST-MEK2, GST-ERK2(K/N) and their mutants were expressed in E. coli DH5α by adding IPTG (0.5 μM) and glutathione- It was purified using Sepharose beads (GE healthcare). Phosphorylated MEK1 was purified from E. coli co-expressing GST-MEK1 and 6xHis-BRaf(V600E). His-MEK1 and MEK2 were purified on a His-Trap HP column using FPLC AKTA system (GE healthcare).

Immunoblotting analysis and antibody immunoblotting analysis were performed as previously described (Takekawa et al., Mol Cell 18, 295-306 2005).
The antibodies used were: polyclonal anti-ERK1 antibody, anti-MEK1 C-18 antibody, anti-C-Raf-1 antibody, monoclonal anti-HA F-7 antibody, anti-GST B-14 antibody, anti-Myc 9E10 antibody, anti-TFPI2 antibody (Santa Cruz); anti-HA mAb 3F10 antibody (Roche); polyclonal anti-phospho-Raf-1 (S338) antibody, anti-phospho-MEK1/2 antibody, anti-phospho -ERK1/2 antibody, anti-GDF15 antibody, anti-S6 antibody and monoclonal anti-phospho-S6 (Cell Signaling Technology); polyclonal anti-COL14A1 antibody, polyclonal anti-PHLDA1 antibody, anti-PHLDA2 (Abcam) antibody; polyclonal anti -TM4SF1 antibody, anti-TM4SF19 antibody, anti-c11orf96 antibody, anti-EMP1 antibody (Sigma); monoclonal anti-Actin antibody (Lab Vision).
All antibodies were diluted 1:1,000 and used for immunoblotting.

Immunofluorescence Microscopy HEK293 cells were cultured on coverslips and transiently transfected with HA-MEK1 or its mutants. Eighteen hours after transfection, cells were fixed in PBS (pH 7.4) containing 3% paraformaldehyde for 10 minutes. The cells were washed with PBS, incubated with 0.1% Triton X-100 for 5 minutes for membrane permeabilization, washed again, and treated with BlockAce (Yukijirushi) for 1 hour at room temperature. Cells were then incubated with 1 μg/ml anti-HA mAb 16B12 (Covance) in PBS containing 2% BSA for 50 minutes at room temperature. Cells were then washed 4 times with PBS and then incubated with Alexa-Fluor 488 goat anti-mouse IgG1 (Molecular Probe) at room temperature for an additional 30 minutes. After incubation, cells were washed four times with PBS and mounted in FluorSave (Calbiochem). Nuclei were visualized by staining with DAPI (0.025 mg/ml in PBS).

Size Exclusion Chromatography An extract of HEK293 cells transiently expressing HA-MEK1 (WT or K57N) was fractionated with a Superdex 75 column (GE healthcare). Eluted fractions were separated by SDS-PAGE, and HA-MEK1 protein was detected with an anti-HA antibody. The GST-tag fused to the N-terminus of recombinant MEK1(WT) and MEK1(K57N) was cleaved using PreScission Protease (Roche), and the MEK1 protein was fractionated with a Superdex 75 column. The eluted fraction was subjected to detection of MEK1 protein with an anti-MEK1 antibody.

Co-immunoprecipitation assay HEK293 cells were transiently transfected with the plasmid and lysed with buffer C after 48 hours. The cell lysate was pretreated with protein G at 4°C for 1 hour and then incubated with an anti-Flag antibody at 4°C for 3 hours. Immunoprecipitates were collected with protein G sepharose and washed with cold buffer C several times. Proteins were spun on SDS-PAGE and subjected to immunoblotting analysis with anti-Myc antibody.

Cell Proliferation and Anchorage-Independent Proliferation Assay For the MEF proliferation assay, cells were plated in 3 wells at 1 x 10 ³ cells/well in DMEM containing 10% FBS, and the cell count was determined using the Cell Counting Kit. Counted at -8 (Dojindo).
For the anchorage-independent growth assay in soft agar, the dishes were previously covered with SeaPlaque Agarose (DMEM containing 0.5% agar and 10% FBS) and then covered with 0.35% MEF cells. Agar (containing 10% FBS) was added. Medium was added on top to prevent the agar from drying out. Specifically, for cells stably expressing HA-MEK1 or its variants (F53S, Q56P, K57N, C121S, Y130C or S218D/S222D), MEF stably expressing cells, 3 x ¹⁰⁴ cells in agar Colonies (>0.1 mm) were counted after inoculation and 4 weeks of culture. Mean colony numbers were calculated based on values obtained from triplicate assays.

Total RNA was purified from each HEK293 cell line using DNA microarray assay miRNeasy kit (Qiagen) and reverse transcribed using PrimeScript RT Master Mix (Takara Bio, Japan). After confirming the purity of the RNA samples, microarray analysis was performed using the Agilent SurePrint G3 Human GE microarray 8x60K v2 kit (Agilent). The mean expression level of each gene was calculated based on data obtained from three independent experiments. The relative increase/decrease of genes was expressed as a fold of the gene expression level in cells expressing the mutant relative to the gene expression level in MEK1(WT)-expressing cells. For the enriched KEGG pathway analysis of MEK1(F53S or K57N)-expressing cells, genes with expression levels >2 or 0.5< compared to the gene expression levels in MEK1(WT)-expressing cells were identified in the DAVID database (http://www.daviddatabase.org/). (http://david.abcc.ncifcrf.gov) and subjected to functional annotation analysis.

Quantitative analysis of mRNA expression levels (q-PCR)
cDNA samples derived from the HEK293 stable cell line were subjected to q-PCR analysis using the Takara Thermal Cycler Dice real time system (Takara Bio, Japan) and Thunderbird SYBR qPCR mix (Toyobo, Japan). Relative gene expression levels were normalized to GAPDH. All q-PCR experiments were performed in triplicate and represent the mean±SEM. Primers used in q-PCR analysis are shown in Tables 1 and 2.

Statistical significance between mean values was tested by Student's t-test. (**, P <0.01; *, P < 0.05). Data are shown as mean±SEM. For comparison of immunostaining between normal and malignant tissues, differential expression was evaluated with the chi-square test. (***, P <0.001; **, P <0.01; *, P < 0.05)

2. result
Activation of MEK mutants from cancer and Ras/MAPK syndrome Previously, an intramolecular regulatory helix (helix-A) located in the N-terminal part of MEK stabilized the structure of the kinase domain, thereby suppressing its activity. (Fischmann et al., Biochemistry 48, 2661-2674 2009). Therefore, we investigated how mutations in and near helix-A, which are frequently found in MEK1/2 derived from cancer and Ras/MAPK syndrome, affect MEK kinase activity.
HEK293 cells were transfected with Myc-ERK2 along with HA-MEK1 (WT; wild-type) or MEK mutants, and ERK2 phosphorylation levels were measured. The MEK variants used here are those from cancer or RAS/MAPK syndrome. All tested MEK mutants showed higher kinase activity than wild-type MEK (Fig. 1A), but the strength of the activity differed depending on the position of the MEK mutation. That is, cancer-derived MEK mutants (e.g., Q56P and K57N) are "highly active" with strong kinase activity, and Ras/MAPK syndrome-derived MEK mutants (e.g., F53S, T55P, D67N and Y130C) , was a "moderately active" type with weaker kinase activity than cancer-derived MEK mutants.

It is known that MEK activity is normally induced by phosphorylation of two serine residues (Ser218 and Ser222) within the T-loop (Alessi et al., EMBO J 13, 1610-1619 1994). However, Ras/MAPK syndrome-derived MEK mutants did not show increased T-loop phosphorylation in E. coli and human cells (FIGS. 1B and C). Similar results are obtained from analysis of MEK2 mutants. To confirm whether the activity of these Ras/MAPK syndrome-derived MEK mutants requires phosphorylation of the T-loop, we investigated non-phosphorylatable MEK mutants in which the T-loop amino acid residue was changed to alanine. A body (MEK1(AA)) was prepared. In vivo kinase assays with these mutants showed that MEK1(AA) with mutations from Ras/MAPK syndrome (F53S or T55P) had kinase activity similar to that of phosphorylatable MEK mutants. (Fig. 1D). These data suggested that MEK mutations from the Ras/MAPK syndrome induced constitutive activation of MEK regardless of the presence or absence of T-loop phosphorylation.

In cancer-derived MEK1 mutants, it was confirmed that Ser218 and Ser222 in the T-loop were strongly phosphorylated (Fig. 1B).
The T-loop of the cancer-derived MEK mutant was highly phosphorylated even when expressed in E. coli (Fig. 1C). Since E. coli does not possess a serine/threonine kinase that uses MEK as a substrate, cancer-derived MEK mutations were thought to autophosphorylate the MEK T-loop. Indeed, cancer-derived MEK mutants (Q56P or K57N) that also carry mutations that lack kinase activity were unable to induce T-loop phosphorylation in vivo and in vitro (FIGS. 2A and B). Also, although the non-phosphorylated wild-type MEK1(AA) mutant is able to phosphorylate ERK in vivo, its kinase activity is lower than that of non-phosphorylated MEK mutants with the Q56P or K57N mutation (MEK1(Q56P+)). AA) and MEK1 (K57N+AA)), it decreased to about 50% (Fig. 2C).
These data suggest that cancer-derived MEK mutations induce potent kinase activity through T-loop autophosphorylation and enhanced phosphorylation-independent basal catalytic activity.

Next, we examined whether this MEK autophosphorylation is due to an intramolecular mechanism or an intermolecular mechanism.
A MEK(Q56P) mutant having kinase activity and a MEK(Q56P) mutant lacking kinase activity as its substrate were mixed, and an in vitro kinase assay was performed. As a result, phosphorylation of the T-loop of MEK1(Q56P), which has lost its kinase activity, could not be detected despite the coexistence of MEK1(Q56P) mutants with kinase activity (Fig. 2D). ). Also, the MEK1(Q56P) mutant, which lacks kinase activity, was unable to phosphorylate other MEK1 in HEK293 cells (Fig. 2E).
We conclude that cancer-induced MEK mutation-induced T-loop autophosphorylation is intramolecular.

Since Gln56 of MEK1 is conserved as Gln60 in MEK2, it is possible that these conserved amino acid substitutions in MEK2, namely cancer-derived MEK2(Q60P) mutants, induce T-loop autophosphorylation. As expected, the MEK2(Q60P) mutant showed autophosphorylation activity as well as the MEK1(Q56P) mutant (FIGS. 3A and B).

Effect of MEK mutations on ERK signaling Activated ERK translocates to the nucleus and phosphorylates various targets to induce various early response genes such as c-jun, c-fos and Egr1 ( Buchwalter et al., Gene 324, 1-14 2004; Pagel and Deindl Indian J Biochem Biophys 48, 226-235 2011). These transcription factors control the expression of specific genes that are essential for a wide variety of intracellular processes such as cell proliferation, differentiation and survival. To investigate the effects of MEK mutations on ERK signaling, cells expressing MEK1(WT) or MEK1 mutants were treated with EGF, and intracellular phosphorylation levels of ERK were detected. In MEK1(WT)-expressing cells, phosphorylation levels of endogenous ERK and its downstream S6 ribosomal protein peaked 2 hours after EGF stimulation and then gradually decreased (Fig. 4A, "P- ERK” and “P-S6K”).
On the other hand, for Ras/MAPK syndrome-derived MEK1(F53S), the phosphorylation pattern of MEK1(F53S) was similar to that of MEK1(WT), but ERK after EGF stimulation by MEK1(F53S) and S6 remained phosphorylated for a long time (FIG. 4A, MEK1(F53S)).
Moreover, in cells expressing cancer-derived MEK1(K57N), constitutive phosphorylation of MEK, ERK and S6 was induced even without EGF stimulation (Fig. 4A, 0 hr of MEK1(K57N)).

Next, we generated HEK293 cells stably expressing ERK-GFP and HA-MEK1 or its mutants, and observed nuclear translocation of ERK in response to EGF stimulation using a time-lapse microscopy system.
Expression of Ras/MAPK syndrome-derived MEK1(F53S) resulted in EGF-dependent ERK translocation to the nucleus and sustained nuclear localization. On the other hand, in MEK1(K57N)-expressing cells, accumulation of ERK in the nucleus was confirmed independently of EGF treatment (Fig. 4B).
Furthermore, the expression of Egr1 (one of the IEGs) in MEK1(WT)-expressing cells depended on the time of EGF treatment, peaking at 5 hours after treatment and then decreasing, whereas MEK1(F53S) expression Its expression was prolonged in cells, and MEK1(K57N)-expressing cells exhibited constitutive expression independent of EGF stimulation (Fig. 4C).
These results suggest that Ras/MAPK syndrome-derived MEK1 mutants and cancer-derived MEK1 mutants induce ERK signals that are different from wild-type MEK1, and may also affect the expression levels of ERK downstream genes. was suggested.

Comparison of gene expression patterns in wild-type MEK1-expressing cells and Ras/MAPK syndrome-derived MEK1 mutant-expressing cells Gene expression patterns indicate that Col14A1 (collagen type 14A1) is a Ras/MAPK syndrome-derived MEK1 with moderate constitutive activity. We found that it was specifically upregulated only when (F53S) was expressed (Fig. 5). Indeed, no upregulation of Col14A1 was observed when the highly active cancer-derived MEK1(K57N) mutant was expressed. Col14A1 is thought to act as a bridging molecule between extracellular fibers and matrix components (Eyre et al., Biochem Soc Trans 30, 844-848 2002; Schuppan et al., J Biol Chem 265, 8823 -8832 1990). In addition, in Ras/MAPK syndrome, it has been reported that collagen fiber deposition is observed in various organs (Hinek et al. Am J Med Genet A 133A, 1-12. 2005; Mori et al., Am J Med Genet 61, 304-309 1996 ), suggesting that expression of genes regulated by Ras/MAPK syndrome-derived MEK1 mutants, mutants that induce constitutive activation of MEK with or without T-loop phosphorylation, It is suggested that it is associated with the onset of Ras/MAPK syndrome.

Comparison of gene expression patterns in wild-type MEK1-expressing cells and cancer-derived MEK1-expressing cells. Gene expression patterns in cells were compared. Expression of the cancer-derived MEK1(K57N) mutant increased or decreased the expression of a number of genes that differed from wild-type MEK1 expression.
Genes whose expression was confirmed to be increased in cancer-derived MEK1(K57N)-expressing cells included MMP1, TM4SF1, etc., which are reported to be expressed in human cancer (Fig. 6). Proteins, which are the gene products of these genes, are actually expressed in various cancer cells (Fig. 7). It was almost completely suppressed by inhibition (Fig. 7, "MEK inhibitor (U0126)"). Furthermore, we found that c11orf96 protein, one of the gene products induced by ERK signaling activation by MEK1(K57N), was significantly expressed in colon, lung and pancreatic tumors compared to normal tissues. , was confirmed by immunohistochemical analysis using TMA (tissue microarrays) (Fig. 8).

The present invention enables early detection of diseases such as cancer and Ras/MAPK syndrome, and is expected to be used in the medical field.

Claims (13)

1. A cancer detection marker comprising at least one molecule selected from a group of proteins whose expression is induced by ERK1 or ERK2, wherein the ERK1 and ERK2 are MEK1 mutations activated by autophosphorylation of the T-loop region A marker for cancer detection characterized in that it is activated by a mutant or a MEK2 mutant.
2. The marker for cancer detection according to claim 1, wherein the mutation in the MEK1 mutant is Q56P, K57N, C121S or E203K.
3. The marker for cancer detection according to claim 1, wherein the mutation in the MEK2 mutant is Q60P.
4. MMP10, EMP1, Rheb2, TM4SF1, TM4SF19, TMEM158, ENDOD1, c2orf89, SLC20A1, LY6K, PLAUR(CD87), PVR(CD155), IL7R(CD127), IL1R2(CD121b), IL4R, TweakR(CD266), CD3D, CD44 , SEMA7, IL13RA2, THBD, XAGE1, PRR9, TRIB1, IER3, c11orf96, c8orf4, PHLDA1, PHLDA2, DUSP5, DUSP6, ERRFI1, GADD45B, IER3, IRX4, SPANXN3, SPANXN4, SPANXN5, TGFb1, BMP2, TFPI2, GDF15, PAEP , CCL7, IL11 and CRLF.
5. The cancer detection marker according to claim 4, comprising at least one protein selected from the group consisting of EMP1, TM4SF1, TM4SF19, c11orf96, PHLDA1, PHLDA2, TFPI2, Rheb2 and GDF15.
6. MMP10, EMP1, Rheb2, TM4SF1, TM4SF19, TMEM158, ENDOD1, c2orf89, SLC20A1, LY6K, PLAUR(CD87), PVR(CD155), IL7R(CD127), IL1R2(CD121b), IL4R, TweakR(CD266), CD3D, CD44 , SEMA7, IL13RA2, THBD, XAGE1, PRR9, TRIB1, IER3, c11orf96, c8orf4, PHLDA1, PHLDA2, DUSP5, DUSP6, ERRFI1, GADD45B, IER3, IRX4, SPANXN3, SPANXN4, SPANXN5, TGFb1, BMP2, TFPI2, GDF15, PAEP , CCL7, IL11 or CRLF antibodies or aptamers for cancer detection.
7. A method for diagnosing cancer or a diagnostic aid for cancer, comprising the step of measuring the expression level of the cancer detection marker according to any one of claims 1 to 6 present in a sample derived from a subject. Method.
8. A method for screening cancer detection markers, comprising the step of searching for a protein that induces the expression of ERK1 or ERK2 that is activated by a MEK1 mutant or MEK2 mutant that is activated by autophosphorylation of the T-loop region .
9. A marker for detecting Ras/MAPK syndrome containing at least one molecule selected from a group of proteins whose expression is induced by ERK1 or ERK2, wherein said ERK1 and ERK2 are active even if the T-loop region is not phosphorylated A marker for detecting Ras/MAPK syndrome characterized by being activated by a MEK1 mutant or MEK2 mutant having
10. 10. The MEK1 mutant of claim 9, wherein the mutations in the MEK1 mutant are F53S, T55P, D67N, P124L, P124Q, G128V, G128N, Y130C, Y130N, Y130H, or E203Q mutations. Ras/MAPK syndrome detection marker.
11. The Ras/MAPK syndrome detection marker according to claim 9, wherein the mutation in the MEK2 mutant is F53C, F53V, F57L, K61E, A62P, P128R, G132V, T134C, or Y134H.
12. Including the step of measuring the expression level of the marker for detecting Ras / MAPK syndrome according to any one of claims 9 to 11 present in a sample derived from a subject, a method for diagnosing Ras / MAPK syndrome or Ras A diagnostic aid for /MAPK syndrome.
13. For detecting Ras/MAPK syndrome, including the step of searching for proteins that induce the expression of ERK1 or ERK2 activated by MEK1 or MEK2 mutants that are active even if the T-loop region is not phosphorylated Marker screening method.
    
    

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