JP6555511B2 - Use of early lung adenocarcinoma malignancy diagnostic markers focusing on epithelial-mesenchymal transition - Google Patents

Description

  The present invention relates to the use of an early lung adenocarcinoma malignancy diagnostic marker focusing on epithelial-mesenchymal transition, and more specifically, an inspection method using an early lung adenocarcinoma malignancy diagnostic marker focusing on epithelial-mesenchymal transition And the kit.

  Lung adenocarcinoma is generally known as a highly malignant cancer because it easily metastasizes and recurs, and 20% of patients with stage I lung adenocarcinoma recur even after surgical resection. Therefore, the group needs postoperative therapy to prevent recurrence and metastasis. However, it is not a necessary treatment for all patients with stage I lung adenocarcinoma. Therefore, if the risk of recurrence / metastasis can be predicted using resected cancer tissue, only appropriate postoperative therapy can be given to patients with poor prognosis, while unnecessary treatment is given to patients with good prognosis. Risk can be reduced. In addition to diagnostic imaging such as X-ray CT and MRI for cancer diagnosis, PET (Positron Emission Tomography) using 18F (fluorine-18) -labeled fluorodeoxyglucose (18F-FDG), specific for specific cancers There are used a method of detecting a cancer marker expressed in the tissue by tissue immunostaining and a method of detecting a cancer marker leaking into blood or tissue. Patents relating to prognostic markers for lung adenocarcinoma include methods for measuring esRAGE (Endogenous Secretory Receptor for Advanced Glycation End products) expression in lung cancer surgical tissues (Patent No. 4779115: Patent Document 1), cells of stage I lung adenocarcinoma There is a method for measuring the mRNA copy number of ACTN4 (WO2011122634 A1: Patent Document 2).

  It is difficult to obtain accurate information for diagnosing the possibility of cancer metastasis or recurrence using conventional chest radiographs, CT, and MRI. A PET test is a dose that does not affect the human body, but it is exposed to radiation because it is injected with a radiation-examined test drug, so pregnant women and breastfeeding women cannot receive the test. Tissue immunostaining requires highly specific antibodies suitable for fixed tissue specimens, as well as antigenic attenuation and inactivation / disappearance at the stage of tissue fixation, and differences in antigen reactivity due to fixation unevenness In some cases, accurate diagnosis may not be possible. In particular, it is difficult to detect phosphorylated proteins by immunostaining. CEA, CYFRA21-1, and Pro-GRP are molecular markers used for lung cancer diagnosis, but they are not sufficient as prognostic prediction markers in addition to insufficient sensitivity in early stage cancer. In addition, there are no diagnostic agents on the market for prognosis diagnosis using prognostic markers.

Patent No. 4779115 WO2011122634 A1

  An object of the present invention is to provide a test method using a malignancy diagnostic marker effective for predicting recurrence and metastasis of early lung adenocarcinoma and a kit thereof.

  Another object of the present invention is to provide an antibody that can be used in a test using a malignancy diagnostic marker that is effective in predicting recurrence and metastasis of early lung adenocarcinoma.

  Another object of the present invention is to provide an internal standard peptide that can be used for examination using a malignancy diagnostic marker that is effective in predicting recurrence and metastasis of early lung adenocarcinoma.

  The present inventors examined predicting prognosis in patients with stage I lung adenocarcinoma using as an index whether the cancer tissue contains cells that are likely to relapse or metastasize. In the case of cancer tissues containing such cells, the possibility of recurrence / metastasis is very high, and by establishing a method for detecting such cells, the prognosis of stage I lung adenocarcinoma cases can be improved. Can be predicted accurately. Specifically, phosphorylation of a protein (hepatocyte growth factor receptor) that is up-regulated in epithelial-mesenchymal transition (EMT) -induced cells involved in cancer malignancy, particularly invasion and metastasis of cancer cells [ Hepatocyte growth factor receptor, c-Met] 1234/1235 tyrosine, NT3 receptor tyrosine kinase [Neurotrophic Tyrosine Kinase Receptor, TrkC] 516th tyrosine, tensin-1 [tensin 1, TNS1] 1404th tyrosine Diagnosis using excised cancer tissue using as a diagnostic marker the phosphorylation of the 92nd serine of sperm-specific antigen 2 [Sperm-specific antigen 2, SSFA2]. The diagnostic method uses a protein extracted from a tissue sample, and the expression level of a diagnostic marker is determined by immunoblot analysis using a phosphorylation specific antibody or multiple reaction monitoring (MRM) analysis using a mass spectrometer I found. Furthermore, an antibody for detecting SSFA2 phosphorylated at the 92nd serine was prepared by synthesis of an internal standard peptide required for multiple reaction monitoring (MRM) analysis and immunoblot analysis. As a result, the prognosis of stage I lung adenocarcinoma can be predicted more easily and with high sensitivity. In patients with stage I lung adenocarcinoma, surgically resected cancer tissues of 9 cases with poor prognosis (group of patients who relapsed within 5 years after surgery) and 9 cases with good outcome (no recurrence within 5 years after surgery) As a result of multiple reaction monitoring (MRM) analysis, c-Met 1234/1235 tyrosine phosphorylation, TrkC 516 tyrosine phosphorylation, TNS1 1404 tyrosine phosphorylation, A statistically significant difference (p <0.0001) was observed in the expression level between the groups with good prognosis. In addition, regarding t-phosphorylation of 1234/1235 of c-Met and 516 tyrosine phosphorylation of TrkC, the difference in the expression level can also be confirmed by immunoblot analysis using each phosphorylation site-specific antibody. did it. On the other hand, regarding the phosphorylation of the 92nd serine of SSFA2, the results of multiple reaction monitoring (MRM) analysis using surgically resected cancer tissues in 20 cases with poor prognosis and 31 cases with good prognosis in stage I lung adenocarcinoma cases There was a statistically significant difference (p <0.005) in the expression level between the poor prognosis group and the good prognosis group, and the expression level was also determined by immunoblot analysis using a specific antibody against the phosphorylation of the 92nd serine of SSFA2. We were able to confirm the difference. SSFA2 was expressed only in the cancer part of poor prognosis and good prognosis groups. The present invention has been completed based on these findings.

The gist of the present invention is as follows.
(1) Detecting phosphorylation of at least one site selected from the group consisting of TrkC at 516 tyrosine, TNS1 at 1404 tyrosine, and SSFA2 at 92th serine for the subject-derived specimen Including early lung adenocarcinoma malignancy test.
(2) If the phosphorylation of at least one site selected from the group consisting of TrkC 516th tyrosine, TNS1 1404th tyrosine, SSFA2 92th serine is enhanced, early lung adenocarcinoma The method according to (1), wherein it is determined that the possibility of metastasis and / or recurrence is high.
(3) The method according to (1) or (2), further comprising detecting phosphorylation of one or both of tyrosine at 1234/1235 of c-Met.
(4) When the phosphorylation of one or both tyrosine at 1234/1235 of c-Met is increased, it is determined that there is a high possibility that early lung adenocarcinoma will metastasize and / or recur (3) the method of.
(5) The method according to any one of (1) to (4), wherein the subject-derived specimen is lung adenocarcinoma tissue, serum, whole blood, or urine.
(6) A group consisting of an antibody against TrkC in which the 516th tyrosine of TrkC is phosphorylated, an antibody against TNS1 in which the 1404th tyrosine of TNS1 is phosphorylated, and an antibody against SSFA2 in which the 92nd serine of SSFA2 is phosphorylated The method according to any one of (1) to (5), wherein the expression level of the phosphorylated protein is measured by an antigen-antibody reaction using at least one antibody selected from the above.
(7) Furthermore, an antigen-antibody reaction using an antibody against c-Met phosphorylated on the 1234th tyrosine of c-Met and / or an antibody against c-Met phosphorylated on the 1235th tyrosine of c-Met The method according to (6), wherein the expression level of phosphorylated c-Met is measured by
(8) Phosphorylation of at least one site selected from the group consisting of 516th tyrosine of TrkC, 1404th tyrosine of TNS1 and 92nd serine of SSFA2 is detected by multiple reaction monitoring (MRM) analysis ( The method according to any one of 1) to (5).
(9) The method according to (8), wherein phosphorylation of one or both of the 1234 / 1235th tyrosine of c-Met is further detected by multiple reaction monitoring (MRM) analysis.
(10) The method according to (9), wherein a multiple reaction monitoring (MRM) analysis is performed under the conditions shown in the following table.

(11) The method according to any one of (1) to (10), wherein the inspection is performed before and / or after the operation.
(12) Selected from the group consisting of a reagent capable of detecting phosphorylation of the 516th tyrosine of TrkC, a reagent capable of detecting the phosphorylation of the 1404th tyrosine of TNS1, and a reagent capable of detecting the phosphorylation of the 92nd serine of SSFA2. An early lung adenocarcinoma malignancy test kit comprising at least one reagent that is used.
(13) The kit according to (12), further comprising a reagent capable of detecting phosphorylation of the 1234th and / or 1235th tyrosine of c-Met.
(14) The kit according to (12) or (13), wherein the reagent is an antibody.
(15) An antibody against SSFA2 in which the 92nd serine of SSFA2 is phosphorylated, and obtained using C + TPLGA (pS) LDEQS as an immunogen.
(16) A peptide consisting of an amino acid sequence of TPLGA (pS) LDEQS to which Cys may be added to the N-terminus.
(17) 7 th tyrosine phosphorylation and / or ¹³ C _6, ¹⁵ N ₂ isotopically labeled which may be AGSLPNYATINGK, seventh tyrosine phosphorylation and / or ¹³ C _6, ¹⁵ N ₂ isotopically labeled DMYDKEYYSVHNK, which may be phosphorylated, IPVIENPQYFR in which the ninth tyrosine is phosphorylated and / or ¹³ C ₆ , ¹⁵ N ₄ isotope-labeled, and the sixth serine is phosphorylated and / or ¹³ C ₆ , ¹⁵ N _A peptide comprising an amino acid sequence selected from the group consisting of TPLGASLDEQSSSTLK, which may be labeled with _two isotopes.
(18) A peptide comprising an amino acid sequence selected from the group consisting of AGSLPNYATINGK, DMYDKEYYSVHNK, IPVIENPQYFR, and TPLGASLDEQSSSTLK, wherein any amino acid in the amino acid sequence may be phosphorylated and / or labeled.
(19) A method for examining lung adenocarcinoma, comprising measuring the level of SSFA2 for a subject-derived specimen.
(20) For SSFA2, when the level in the subject-derived specimen is high, it is determined that there is a high possibility of having lung adenocarcinoma, and when the level is low, the patient may be suffering from lung adenocarcinoma The method according to (19), wherein it is determined that the property is low.
(21) The subject is a patient undergoing treatment for lung adenocarcinoma, and the level of SSFA2 in the subject-derived specimen is measured several times at different times, and when the level decreases, lung adenocarcinoma is treated. (19) The method according to (19), wherein it is determined that the patient has recovered from lung cancer, and when the level does not decrease, it is determined that treatment has not recovered from lung adenocarcinoma or recovery is insufficient.
(22) The method according to any one of (19) to (21), wherein the subject-derived specimen is cancer tissue, serum, whole blood, or urine.
(23) A test kit for lung adenocarcinoma comprising a reagent capable of specifically detecting SSFA2.
(24) The kit according to (23), wherein the reagent is an antibody.

  The present invention is effective for early stage lung adenocarcinoma, particularly stage I (stage IA and stage IB) non-small cell lung cancer.

  According to the present invention, a malignancy screening test for prognosis prediction can be performed accurately and with high sensitivity for patients with early lung adenocarcinoma.

The amount of E-cadherin and vimentin in cell lysates was examined by EMT-induced immunoblot analysis in A549 cells treated with TGF-β, but the expression level of β-actin was the same, but E-cadherin in response to TGF-β treatment. It was confirmed that the amount decreased and the vimentin amount increased (A). As a result of examining morphological changes, it was confirmed that A549 cells changed from epithelial cell-like to spindle-like fibroblast-like by TGF-β treatment (B). TGF-β (-): TGF-β non-treated cells, TGF-β (+): TGF-β treated cells The amount of each protein in cells treated with TGF-β As a result of immunoblot analysis using each specific antibody, the amount of TrkC, TNS1, and c-Met proteins in the cells was not changed by TGF-β treatment. It was confirmed that the amount of TrkC phosphorylated by the 1st tyrosine and the amount of c-Met phosphorylated by the 1234th and / or 1235th tyrosine increased. TGF-β (-): TGF-β non-treated cells, TGF-β (+): TGF-β treated cells In the TGF-β-treated cells, the amount of each peptide amount TNS1 in which the 1404th tyrosine was phosphorylated (A), the amount of peptide in which the 1234 / 1235th tyrosine of c-Met was phosphorylated (B), The amount of peptide (C) phosphorylated at the 516th tyrosine of TrkC is a statistically significant difference between TGF-β-untreated cells and TGF-β-treated cells as a result of multiple reaction monitoring (MRM) analysis. (P <0.001). On the other hand, there was no statistically significant difference in the amount of these non-phosphorylated peptides in the dephosphorylated sample (D, E, F). TGF-β (-): TGF-β non-treated cells, TGF-β (+): TGF-β treated cells Multiple reaction monitoring (MRM) using stage I lung adenocarcinoma surgically resected cancer tissue in 9 cases with poor prognosis and 9 cases with good prognosis As a result of analysis, the amount of peptide in which 1404 tyrosine of TNS1 was phosphorylated (A left), the amount of peptide in which tyrosine 1234/1235 of c-Met was phosphorylated (B left), 516 tyrosine of TrkC In all cases, there was a statistically significant difference (p <0.0001) between the good prognosis group and the poor prognosis group. On the other hand, there was no statistically significant difference in the amount of these non-phosphorylated peptides in the dephosphorylated sample (D left, E left, F left). In addition, the amount of TrkC, TNS1, and c-Met proteins in the tissue was not changed by immunoblot analysis (D right, E right, F right), but c-phosphorylated at the 1234th and / or 1235th tyrosine. It was confirmed that the amount of Met (B right) and the amount of TrkC phosphorylated at the 516th tyrosine (C right) increased. GP: good prognosis group, PP: poor prognosis group, Y [Pho]: phosphorylated tyrosine Three types of phosphopeptides obtained by multiple reaction monitoring (MRM) analysis were classified as above average and below according to 9 cases with poor prognosis and 9 cases with good prognosis of relapse-free survival stage I lung adenocarcinoma In this case, it was confirmed that the survival period after the operation was statistically significantly shorter than the average value group (p <0.0001, Kaplan-Meier method). Phospho-Low: a group with a phosphorylated peptide amount below the average value, Phospho-High: a group with a phosphorylated peptide amount above the average value Receiver operating characteristics (ROC) curve Y stage 9 lung adenocarcinoma group with poor prognosis group and 9 cases with good prognosis group based on 3 types of phosphopeptides obtained by multiple reaction monitoring (MRM) analysis A graph was drawn using sensitivity on the axis and specificity on the x-axis, indicating that phosphorylation of these proteins is useful as a diagnostic marker. GP: good prognosis group, PP: poor prognosis group, AUC: area under the curve (ROC curve area) Dephosphorylation efficiency by alkaline phosphatase Antiphosphorylated tyrosine antibody (PY20) (Santa Cruz Biotechnology) is used to detect intracellular phosphorylated proteins before and after dephosphorylation, and many proteins are dephosphorylated It was confirmed. CIAP +: Cell extract protein that has been dephosphorylated CIAP-: Cell extract protein that has not been dephosphorylated Measurement of the amount of each protein in cells treated with TGF-β. As a result of immunoblot analysis using each specific antibody, it was confirmed that the amount of SSFA2 protein in cells was not changed by TGF-β treatment, but the amount of SSFA2 phosphorylated by the 92nd serine increased. TGF-β (-): TGF-β non-treated cells, TGF-β (+): TGF-β treated cells Immunoblotting analysis using specific antibodies for protein levels in surgically resected cancer tissues of stage I lung adenocarcinoma confirmed that SSFA2 is expressed only in the cancerous part of both poor prognosis group and good prognosis group (A). Furthermore, as a result of examining the amount of protein phosphorylated at the 92nd serine of SSFA2 by immunoblot analysis using a phosphorylation site-specific antibody, it was found in the cancer part of the poor prognosis group compared to the cancer part of the good prognosis group. It was found that the expression level increased (B). In the surgically resected tissue of stage I lung adenocarcinoma, 20 or 17 cases of poor peptide prognosis group, 31 or 32 cases of good prognosis group using multiple resected cancer tissues of stage I lung adenocarcinoma, multiple reactions respectively The amount of phosphorylated peptide or non-phosphorylated peptide was quantified by monitoring (MRM) analysis. As a result, there was a statistically significant difference (p <0.005) between the good prognosis group and the poor prognosis group in the amount of peptide in which the 92nd serine of SSFA2 was phosphorylated (left). On the other hand, there was no statistically significant difference in the amount of these non-phosphorylated peptides in the dephosphorylated sample (right). GP: good prognosis group, PP: poor prognosis group, S [Pho]: phosphorylated serine, N.S .: non-significant Relapse-free survival period In stage I lung adenocarcinoma 20 cases with poor prognosis group and 31 cases with good prognosis group, when the amount of phosphopeptides obtained by multiple reaction monitoring (MRM) analysis is classified as above average or below, the average It was confirmed that the postoperative survival time was statistically significantly shorter in the group above the average than the group below the value (p <0.005, Kaplan-Meier method). Phospho-Low: a group with a phosphorylated peptide amount below the average value, Phospho-High: a group with a phosphorylated peptide amount above the average value Receiver operating characteristics (ROC) curve Sensitivity on the y-axis based on the amount of phosphorylated peptide obtained by multiple reaction monitoring (MRM) analysis in 20 cases with poor prognosis and 31 cases with good prognosis in stage I lung adenocarcinoma (sensitivity), graphed using specificity on the x-axis, showing that phosphorylation of these proteins is useful as a diagnostic marker. GP: good prognosis group, PP: poor prognosis group, AUC: area under the curve (ROC curve area)

  Hereinafter, the present invention will be described in detail.

  The present invention detects phosphorylation of at least one site selected from the group consisting of TrkC at 516 tyrosine, TNS1 at 1404 tyrosine, SSFA2 at 92th serine, from a subject-derived specimen. To provide a malignancy test for early lung adenocarcinoma, including

  When the phosphorylation of at least one site selected from the group consisting of 516th tyrosine of TrkC, 1404th tyrosine of TNS1 and 92nd serine of SSFA2 is enhanced, early lung adenocarcinoma is metastasized and / or Alternatively, it can be determined that the possibility of recurrence is high.

  In the method of the present invention, phosphorylation of one or both tyrosine at the 1234 / 1235th position of c-Met may be detected.

  When phosphorylation of one or both tyrosine at 1234/1235 of c-Met is enhanced, it can be determined that there is a high possibility that early lung adenocarcinoma will metastasize and / or recur.

  Examples of the specimen derived from the subject include lung adenocarcinoma tissue, serum, whole blood, urine and the like.

  TrkC antibody against TrkC phosphorylated at 516th tyrosine, TNS1 antibody against TNS1 phosphorylated at 1404th tyrosine, SSFA2 antibody against SSFA2 phosphorylated at serine 92th And at least one site selected from the group consisting of TrkC at 516 tyrosine, TNS1 at 1404 tyrosine, and SSFA2 at 92th serine. be able to.

  Furthermore, an antigen-antibody reaction using an antibody against c-Met in which the 1234th tyrosine of c-Met was phosphorylated and / or an antibody against c-Met in which the 1235th tyrosine of c-Met was phosphorylated was used to produce phosphorylation. The expression level of oxidized c-Met may be measured.

  Using the antigen-antibody reaction, the expression level of phosphorylated protein can be measured by ELISA or immunoblotting.

  In addition, multiple reaction monitoring (MRM) analysis can detect phosphorylation of at least one site selected from the group consisting of TrkC at 516 tyrosine, TNS1 at 1404 tyrosine, and SSFA2 at 92th serine. it can.

  Furthermore, phosphorylation of one or both tyrosine 1234/1235 of c-Met may be detected by multiple reaction monitoring (MRM) analysis.

In one embodiment of the present invention, multiple reaction monitoring (MRM) analysis can be performed under the conditions shown in the table below.

* Y [Pho] or S [Pho]; represents phosphorylated tyrosine or phosphorylated serine
* [ ¹³ C ₆ , ¹⁵ N ₂ ]; Peptide containing 6 ( ¹³ C) and 2 ( ¹⁵ N) and stable isotope-labeled K (lysine)
* [ ¹³ C ₆ , ¹⁵ N ₄ ]; Peptide containing 6 ( ¹³ C) and 4 ( ¹⁵ N) and stable isotope-labeled R (arginine)

  The examination can be performed before and / or after the operation.

  The present invention is selected from the group consisting of a reagent capable of detecting phosphorylation of the 516th tyrosine of TrkC, a reagent capable of detecting phosphorylation of the 1404th tyrosine of TNS1, and a reagent capable of detecting phosphorylation of the 92nd serine of SSFA2. There is also provided an early lung adenocarcinoma malignancy test kit comprising at least one of the reagents described above.

  The kit of the present invention may further contain a reagent capable of detecting phosphorylation of the 1234th and / or 1235th tyrosine of c-Met.

  The reagent is preferably an antibody, an antibody that specifically binds to TrkC phosphorylated at the 516th tyrosine, an antibody that specifically binds to TNS1 phosphorylated at the 1404th tyrosine, and the serine at the 92nd phosphorylate. An antibody that specifically binds to oxidized SSFA2 or an antibody that specifically binds to c-Met phosphorylated at the 1234th and / or 1235th tyrosine. An antibody that specifically binds to TrkC phosphorylated at the 516th tyrosine, an antibody that specifically binds to TNS1 phosphorylated at the 1404th tyrosine, c that is phosphorylated at the 1234th and / or 1235th tyrosine Antibodies that specifically bind to -Met are commercially available and available. An antibody that specifically binds to SSFA2 phosphorylated at the 92nd serine was inoculated to the animal using C + TPLGA (pS) LDEQS as an immunogen, and then collected from this animal. did. Therefore, the present invention provides an antibody against SSFA2 in which the 92nd serine of SSFA2 is phosphorylated, and obtained using C + TPLGA (pS) LDEQS as an immunogen.

  The antibody may be labeled with a radioisotope, enzyme, luminescent material, fluorescent material, biotin, and the like. In addition, the target molecule (in the present invention, TrkC phosphorylated at the 516th tyrosine, TNS1 phosphorylated at the 1404th tyrosine, SSFA2, 1234 and / or the 1235th tyrosine phosphorylated at the 92nd serine) When the target antibody is detected by reacting the secondary antibody that binds to the primary antibody after the reaction of the primary antibody that specifically binds to phosphorylated c-Met), label the secondary antibody. It is preferable (the primary antibody is not labeled).

  In addition, the kit of the present invention contains standard proteins (TrkC phosphorylated at the 516th tyrosine, TNS1 phosphorylated at the 1404th tyrosine, and the 92nd serine phosphorylated by ELISA and immunoblot analysis). Oxidized SSFA2, 1234 and / or c-Met phosphorylated at 1235th tyrosine), internal standard peptide (for multiple monitoring (MRM) analysis, TrkC partial peptide phosphorylated at 516th tyrosine, A partial peptide of TNS1 phosphorylated at the 1404th tyrosine, a partial peptide of SSFA2 phosphorylated at the 92th serine, a partial peptide of c-Met phosphorylated at the 1234 and / or 1235th tyrosine), A buffer, a substrate (when the antibody is labeled with an enzyme), a reaction stop solution, a washing solution, a reaction vessel, a user manual, and the like may be included.

  Furthermore, the present invention also provides a peptide consisting of the amino acid sequence of TPLGA (pS) LDEQS, to which Cys may be added at the N-terminus. These peptides can be used as an immunogen for obtaining an antibody against SSFA2 in which the 92nd serine of SSFA2 is phosphorylated.

Furthermore, the present invention relates to AGSLPNYATINGK in which the seventh tyrosine may be phosphorylated and / or ¹³ C ₆ , ¹⁵ N ₂ isotope-labeled, and the seventh tyrosine is phosphorylated and / or ¹³ C ₆ , ¹⁵ N DMYDKEYYSVHNK, which may be labeled with ₂ isotopes, IPVIENPQYFR where 9th tyrosine is phosphorylated and / or ¹³ C ₆ , ¹⁵ N ₄ isotope labeled, and 6th serine is phosphorylated and / or ¹³ C Provided is a peptide consisting of an amino acid sequence selected from the group consisting of TPLGASLDEQSSSTLK optionally labeled with ₆ , ¹⁵ N ₂ isotope. Alternatively, the present invention provides a peptide comprising an amino acid sequence selected from the group consisting of AGSLPNYATINGK, DMYDKEYYSVHNK, IPVIENPQYFR and TPLGASLDEQSSSTLK, wherein any amino acid in the amino acid sequence may be phosphorylated and / or labeled To do. These peptides are available as peptides for internal standards in multiplex monitoring (MRM) analysis.
Furthermore, the present invention provides a method for examining lung adenocarcinoma, comprising measuring the level of SSFA2 for a specimen derived from a subject.

  Regarding SSFA2, when the level in the subject-derived specimen is high, it is determined that the possibility of having lung adenocarcinoma is high, and when the level is low, the possibility of having lung adenocarcinoma is low Can be determined. Therefore, the method of the present invention can be used for diagnosis of lung adenocarcinoma (determination of presence or absence of lung adenocarcinoma).

  In addition, if the subject is a patient undergoing treatment for lung adenocarcinoma, the SSFA2 is measured multiple times at different times in the subject-derived specimen, and if the level decreases, the lung adenocarcinoma is treated by treatment. If the level does not decrease, it can be determined that the treatment has not recovered from lung adenocarcinoma or the recovery has been insufficient. Therefore, the method of the present invention can also be used for changes in the pathology of lung adenocarcinoma, current pathological conditions, prognostic examination, and confirmation of the therapeutic effect of lung adenocarcinoma.

  Examples of the subject-derived specimen include cancer tissue, serum, whole blood, urine and the like.

  The present invention also provides a test kit for lung adenocarcinoma comprising a reagent capable of specifically detecting SSFA2.

  The reagent is preferably an antibody, and is preferably an antibody that specifically binds to SSFA2. Such antibodies are commercially available and can be used. The antibody may be either a monoclonal antibody or a polyclonal antibody. The antibody may be labeled with a radioisotope, enzyme, luminescent material, fluorescent material, biotin, and the like. In addition, when a target antibody is detected by reacting a secondary antibody that binds to the primary antibody after the reaction of the primary antibody that specifically binds to the target molecule (SSFA2), the secondary antibody may be labeled. (Primary antibody is not labeled). Using the antigen-antibody reaction, the expression level of SSFA2 can be measured by ELISA or immunoblotting.

In addition, the kit of the present invention may contain a standard protein (SSFA2), a buffer, a substrate (when the antibody is labeled with an enzyme), a reaction stop solution, a washing solution, a reaction vessel, a user manual, and the like.

Hereinafter, the present invention will be described in more detail with reference to examples.
[Example 1]
INTRODUCTION In current cancer medicine, many of them can be detected early and surgically removed to achieve good therapeutic results. However, lung adenocarcinoma, which is generally known as a high-grade cancer because of its easy metastasis and recurrence, and is the leading cause of death from cancer in developed countries, is an early surgical treatment of affected areas from patients with lung adenocarcinoma Even if surgically removed, 20% have relapsed and died within 5 years after ^surgery1,2 . This shows that adjuvant therapy such as chemotherapy is necessary immediately after surgery, even in early cancers, as in more advanced cancers. However, in the remaining 80%, such treatment is not necessary, and development of diagnostic markers that can accurately identify patients at risk of recurrence that require various adjuvant treatments in this situation Is required. So far, in lung adenocarcinoma, using lung adenocarcinoma tissues collected immediately after surgery, we have identified proteins with different expression levels between good prognosis group and poor prognosis group, and have found several prognostic marker marker proteins ^3-5 . However, no research has been conducted to search for prognostic markers by focusing on changes in phosphorylation associated with induction of epithelial-mesenchymal transition (EMT).

EMT is a phenomenon in which epithelial cells lose cell-cell adhesion and transform into mesenchymal-like (non-epithelial) cells, and it is known that TGF-β (β-type mutant growth factor) is involved in its induction ^6-11 . It has been suggested that cells in which EMT has been induced have motility and invasive ability, leading to cancer invasion and metastasis ^6-11 . Therefore, detecting phosphorylated proteins related to EMT induction caused by TGF-β, and discovering molecules that can detect the presence of EMT-induced cells in tumor tissue, are considered EMT induction (invasion and metastasis of cancer cells). It is highly likely to lead to the development of a novel malignancy diagnostic marker that predicts prognosis using easiness).

  Therefore, in this study, we captured specific changes in phosphorylated proteins in EMT-induced cells involved in cancer malignancy, and these correlated with good and poor prognosis in tissue samples from patients with early lung adenocarcinoma. Whether it fluctuated was confirmed by immunoblot analysis and multiple reaction monitoring (MRM) analysis.

Materials and Methods Cell Culture
A549 cells (human lung adenocarcinoma cell line) were fixed at 37 ° C using RPMI-1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% (w / v) fetal bovine serum (FBS). Cultivation was performed in a CO ₂ incubator. TGF-β treatment involves culturing cells in low serum (0.1% FBS) medium for 24 hours, followed by 48 hours in medium supplemented with TGF-β (HumanZyme, Chicago, IL, USA, final concentration 5 ng / mL) This was done by culturing. On the other hand, TGF-β non-treatment was performed by culturing for 48 hours in a medium to which only a solvent (DMSO) in which TGF-β was dissolved was added.

Tissue samples Cancer tissues removed from 18 early stage (Ia or Ib) lung adenocarcinoma patients who agreed to use tissue samples for research, etc., and frozen tissue samples stored at -80 ° C until use in this study Obtained from Kanagawa Cancer Center (Table 1). The breakdown consists of 9 patients with poor prognosis who relapsed within 5 years after excision and 9 patients with good prognosis who did not relapse within 5 years after surgery.

Protein preparation Collected cells or tissues using Sample Grinding Kit (GE Healthcare, Piscataway, NJ, USA), 8 M urea, 4% (w / v) sodium deoxycholate, Protease Inhibitor Mix (GE Healthcare) And disrupted in 50 mM ammonium bicarbonate solution containing Phosphatase Inhibitor Cocktail 2 and 3 (Sigma, Madison, WI, USA). The crushed material was sonicated 5 times (1 second interval) with UR-21P (TOMY, Tokyo, Japan), then centrifuged at 15,000g (4 ° C) for 15 minutes to recover the supernatant, and this was extracted with protein Liquid. The dephosphorylation treatment was carried out by reacting 10 units of alkaline phosphatase (CIAP, Takara Shuzo, Tokyo, Japan) for 1 hour at 37 ° C. with respect to 50 μg of protein.

Immunoblot analysis Protein extracts were separated using SuperSep ^™ Ace (10% T, 17 wells) (Wako Pure Chemicals) and Trans-Blot® Turbo ^™ Transfer System (Bio-Rad Laboratories, Hercules, CA). , USA). After that, PVDF membrane was added to T-PBS (final concentration 10 mM Na ₂ HPO ₄ · 12H ₂ O, 1.8 mM KH ₂ PO ₄ , 0.137 M NaCl, 2.7) containing 5% (w / v) skim milk (Morinaga Milk Industry, Japan). Blocking operation to suppress non-specific reaction was performed by reacting with mM KCl, 0.05% [v / v] Tween20) for 1 hour. For primary antibodies, rabbit anti-tensin 1 antibody (Abcam, MA, USA), rabbit anti-c-Met antibody (MBL, Nagoya, Japan), rabbit anti-phosphorylated c-Met [Tyr1234 / 1235] antibody (Cell Signaling Technology) Rabbit anti-TrkC antibody (R & D, Minneapolis, MN, USA), rabbit anti-phosphorylated TrkC [Tyr516] antibody (EnoGene, New York, NY, USA), mouse anti-E-cadherin antibody (BD Biosciences, San Jose, CA, USA), rabbit anti-vimentin antibody (Cell Signaling Technology), rabbit anti-actin β antibody (H-196, Santa Cruz Biotechnology, Dallas, TX, USA), anti-phosphotyrosine antibody (PY20, Santa Cruz Biotechnology) 5% ( w / v) A 1000-fold diluted solution of T-PBS containing skim milk was used and allowed to react for 12 hours or more. After the reaction, the PVDF membrane is washed with T-PBS, and 5% (w / v) skim milk containing goat anti-rabbit IgG antibody or goat anti-mouse IgG antibody (Santa Cruz Biotechnology) labeled with peroxidase (HRP) as a secondary antibody A 5,000-fold diluted solution with T-PBS was used and allowed to react for 1 hour. After the reaction, the PVDF membrane was washed with T-PBS, and reacted with HCL substrate ECL Plus Western Blotting Detection Reagents (GE Healthcare) for 5 minutes. The signal of each protein was detected using LAS4000mini luminescence image analyzer (GE Healthcare).

Preparation of peptide solution DTT was added to the protein extract so as to have a final concentration of 10 mM, followed by reduction at room temperature for 45 minutes. After the reduction, iodoacetamide was added to a final concentration of 5 mM, and the reaction was carried out at room temperature for 30 minutes to alkylate cysteine residues. Thereafter, the mixture was centrifuged at 10,000 g for 15 minutes to recover the supernatant, and 3 times the amount of 50 mM ammonium bicarbonate was added to dilute the final concentration of urea to 2 M. After protein quantification using NanoDrop 2000c (Thermo Fisher Scientific, Bremen, Germany), trypsin (Sigma) and protein: trypsin (enzyme) ratio of 20: 1 (w / w) and incubated at 37 ° C. for 16 hours. After adding 20% (v / v) TFA of 1/20 of the digest, centrifuge for 5 minutes at 15,000 g (4 ° C), collect the supernatant, and add peptide by phase transfer dissolution method (PTS method) The sodium deoxycholate contained in the solution was removed ¹² . The collected peptide solution was once dried, then redissolved in 0.1% trifluoroacetic acid, desalted using Sep-PakR C18 Plus Short Cartridge (Waters, Milford, MA, USA), and dried. Antigen-antibody reaction using Phospho-Tyrosine Mouse mAb (P-Tyr-100) beads (Cell Signaling Technology, Boston, USA) ¹³ was used for concentration of tyrosine phosphorylated peptide in trypsin digest (1 mg) ¹³ . Thereafter, the recovered peptide was concentrated and desalted using a chip column (C18 Stage Tip) using a C18 filter ¹⁴ . Each peptide was stored at −20 ° C. in a dried state until mass spectrometry.

Peptide quantification by multiple reaction monitoring (MRM) analysis Multiple reaction monitoring (MRM) analysis is performed using the TripleTOF5600 system (AB Sciex, Foster City, CA, USA) coupled with DiNa-AP (KYA Technologies, Tokyo, Japan). It was. The concentration column connected to DiNa-AP is HiQ sil C18W-3, 500 μm id × 1 mm (KYA Technologies), and the separation column is nanoscale HiQ sil C18W-3, 100 μm id × 10 cm (KYA Technologies). The flow rate was 200 nL / min. A buffer contains 0.1% (v / v) formic acid solution containing 2% (v / v) acetonitrile, and B buffer contains 0.1% (v / v) formic acid solution containing 80% (v / v) acetonitrile. Was used. Peptide adsorption is performed with A buffer 98% (v / v) and B buffer 2% (v / v), and elution is continuously increased from 2% to 40% over 42 minutes. Made using the program. Multiple reaction monitoring (MRM) scans are performed in positive mode with ion spray voltage 2300V, curtain gas and spray gas mode set to 20, resolution set to unit, measured mass (m / z) range from 100 to 1,600 Da and MS2 accumulation times were set to 0.1 seconds. Phosphorylated peptides and stable isotope-labeled-phosphorylated peptides were synthesized at Scrum (Tokyo, Japan) (Table 2). MRMPilot v2.0 (AB Sciex) software was used to automatically calculate the precursor ion / fragment ion pair (MRM transition) and collision energy of the target peptide. Finally, reproducible y ions and MRM transitions were selected (Table 2). For each peptide sample, a stable isotope labeled-phosphorylated peptide at a concentration of 25 fmol was added and used as an internal standard for quantification. The calibration curve was prepared using 0.5, 1, 5, 10, and 50 fmol peptides, and the accuracy of the calibration curve was kept within 20% (% CV <20). Mass spectrometry data obtained as a result of mass spectrometry was analyzed using MultiQuant v.2.1 (AB Sciex), and the amount of peptide was calculated.

Statistical analysis
Statistical analysis such as Mann-Whitney test to show statistical significance of differences between two groups, Kaplan-Meier method to perform recurrence-free survival analysis, receiver operating characteristic (ROC) curve analysis, etc. GraphPad prism 5 (V. 5.04, GraphPad Software, San Diego, CA, USA).

Results 1. EMT induction in A549 cells by TGF-β treatment From the results of immunoblot analysis using each protein-specific antibody, the amount of E-cadherin in cultured A549 lung adenocarcinoma cells decreased in response to TGF-β treatment, and the amount of vimentin Was found to increase (FIG. 1A). Further, as a result of examining morphological changes, it was confirmed that A549 cells were changed from epithelial cell-like morphology to spindle-like fibroblast-like morphology by TGF-β treatment (FIG. 1B). These results are the same as the previous ^{reports 7} and ¹⁵ on EMT induction by TGF-β treatment, and it was judged that the A549 cells used in this study were also induced by TGF-β treatment.
2. Detection of tyrosine phosphorylated protein specifically changing in AGF cells treated with TGF-β From the comprehensive relative quantitative analysis of tyrosine phosphorylated peptides using a mass spectrometer, the expression level was significantly increased by TGF-β treatment in A549 cells As a phosphorylated peptide in which TNS1 is increased, a peptide in which the 1404th tyrosine of TNS1 is phosphorylated, a peptide in which the 1234 / 1235th tyrosine of c-Met is phosphorylated, and a peptide in which the 516th tyrosine of TrkC is phosphorylated I found. Immunoblot analysis using specific antibodies against TrkC, TNS1, c-Met and TrkC phosphorylated at 516th tyrosine and antibodies specific to c-Met phosphorylated at 1234 and / or 1235th tyrosine As a result, it was confirmed that the expression level of these proteins in the cells was not changed by TGF-β treatment, but phosphorylation at each site was enhanced (FIG. 2). In addition, by multiplex reaction monitoring (MRM) analysis using a TripleTOF 5600 system as a mass spectrometer, the amount of each phosphorylated peptide in the cell and the amount of non-phosphorylated peptide derived from each dephosphorylated sample (FIG. 7) ( Changes in the total peptide amount were examined (FIG. 3). As a result, the multiple reaction monitoring (MRM) analysis as well as the immunoblot analysis showed no significant difference in the total peptide amount (Fig. 3D, E, F), but TNS1 1404 tyrosine was phosphorylated. For peptide amount (Fig. 3A), c-Met 1234/1235 tyrosine phosphorylated (Fig. 3B), TrkC 516 tyrosine phosphorylated (Fig. 3C), all A statistically significant increase (p <0.001) was confirmed in response to TGF-β treatment.
3. Differences in phosphoprotein levels between postoperative tissues in patients with good prognosis and poor prognosis in early stage (Ia or Ib) lung adenocarcinoma patients Cryopreserved lung adenocarcinoma tissues of 18 patients removed by surgery Mass spectrometer for the amount of phosphorylated protein in the tissues of 9 patients who relapsed lung adenocarcinoma 5 years later (poor prognosis group, PP) and 9 patients who did not relapse (good prognosis group, GP) The reaction was compared by multiple reaction monitoring (MRM) analysis using and immunoblot analysis using each specific antibody (FIG. 4). As a result of multiple reaction monitoring (MRM) analysis, the amount of TNS1 1404 tyrosine phosphorylated (Figure 4A left), c-Met 1234/1235 tyrosine phosphorylated peptide (Figure 4B left) ), The amount of peptide phosphorylated at the 516th tyrosine of TrkC (FIG. 4C left) was all statistically significant (p <0.0001) between the good prognosis group and the poor prognosis group. On the other hand, there was no significant difference in the amount of these non-phosphorylated peptides in the dephosphorylated sample (FIG. 4D left, E left, F left). In addition, the amount of TrkC, TNS1, and c-Met proteins in the tissues did not change by immunoblot analysis using each specific antibody (FIG. 4D right, E right, F right), but the 1234 and / or 1235th protein It was confirmed that the amount of c-Met phosphorylated by tyrosine (FIG. 4B right) and the amount of TrkC phosphorylated by the 516th tyrosine (FIG. 4C right) increased.
Furthermore, based on the results obtained by multiple reaction monitoring (MRM) analysis, a recurrence-free survival curve for each phosphopeptide amount was prepared. As a result, for the 9 cases with poor prognosis and 9 cases with good prognosis in stage I lung adenocarcinoma, the three types of phosphopeptides obtained by multiple reaction monitoring (MRM) analysis were classified as above average and below As a result, it was confirmed that the survival rate after the operation was statistically significantly shorter (p <0.0001) in the group above the average value compared to the group below the average value (FIG. 5). Moreover, the results of receiver operating characteristic (ROC) curve analysis showed that proteins phosphorylated at these sites were useful as diagnostic markers (Fig. 6).

Discussion
EMT is related to cancer invasion and metastasis ^6-11 . When EMT is induced, the protein whose expression level varies is highly likely to be related to the malignancy of cancer. There are examples of quantitative analysis of protein in EMT induction using a mass spectrometer ^15,16 , but examples of investigating protein fluctuations focusing on phosphorylation, which plays an important role in intracellular signal transduction There are few ^17,18 . In this study, in response to TGF-β treatment, we enhanced phosphorylation of the 1404 tyrosine of TNS1, phosphorylation of the 1234/1235 tyrosine of c-Met, and the 516th tyrosine phosphorylation of TrkC. I found out that These tyrosine phosphorylation changes were found to be detectable with high sensitivity and high accuracy by multiple reaction monitoring (MRM) analysis as well as conventional immunoblot analysis using specific antibodies. . Detection of target proteins using antibodies is a highly sensitive and excellent technique, but detection depends on the acquisition of highly specific antibodies, and it takes several months to create new antibodies. is necessary. On the other hand, quantitative analysis by multiple reaction monitoring (MRM) analysis eliminates the need to generate antibodies that specifically recognize modified sites, and allows rapid evaluation of the expression level of post-translationally modified proteins. It is also possible to quantify proteins simultaneously ^19,20 . In addition, the TripleTOF 5600 system used for multiple reaction monitoring (MRM) analysis in this study had good resolution and was suitable for quantitative analysis because it uses a time-of-flight (TOF) mass spectrometer for detection. . Therefore, multiple reaction monitoring (MRM) analysis using the TripleTOF 5600 system is an excellent method for detecting this diagnostic marker.
TNS1, detected in this study as a phosphorylated protein associated with EMT induction, is a cell adhesion molecule that binds to actin filaments and interacts with proteins containing phosphorylated tyrosine ^21,22 . Autophosphorylation of FAK occurs when TNS1 binds to extracellular matrix adhesion proteins via integrins. As a result, by the tyrosine kinase of the Src family phosphorylated tyrosine residues resulting binds, p130Cas ²² which, Shc, paxillin, tyrosine phosphorylation of docking proteins or adapter proteins such as TNS is induced secondarily. Through this cascade of protein tyrosine phosphorylation, the pathway from PI3K to Akt and the pathway from Grb2 / Sos / Ras to MAPK are activated, and cell survival is maintained and the cell cycle proceeds, It has been suggested ^{21, 22} . As for the phosphorylation analysis of TNS1, phosphorylation at many serine / threonine / tyrosine sites has been detected from the analysis using HEK293 cells, but the role of phosphorylation at each site has been clarified. Not ²³ . The 1404th tyrosine detected in this study has also been reported to be phosphorylated, but its function has not been clarified, and is related to EMT induction and early prognosis of lung adenocarcinoma. This is the first time this is shown. TrkC is also known as a neurotrophic factor (neurotrophin) receptor, and TrkA and TrkB are also known for this receptor ²⁴ . From the results of the amino acid sequence homology search between TrkC and TrkA, it was found that the 516th tyrosine, which is the TrkC phosphorylation site detected in this study, corresponds to the 490th tyrosine of TrkA. This phosphorylation of TrkA at the 490th tyrosine has been reported to be involved in cancer growth by interacting with Shc and Frs2 and activating the Ras-MAP kinase cascade ²⁵ . Also, by that or ²⁶ to suppress the breast cancer metastasize to the lungs when suppressed TrkC, TrkC in breast cancer cells form a ETV6 the chimeric tyrosine kinases, which binds directly to type II TGF-beta receptor prevents interaction between the type I TGF-beta receptor, resulting TGF-bata / Smad signaling by suppressing has also been suggested that there is a role in inhibiting tumor suppression activity of TGF-beta ^27. However, this is the first discovery as there is no example of the relationship between EMT induction of phosphorylation at the 516th tyrosine detected in this study and the prognosis of early lung adenocarcinoma. On the other hand, c-Met has already been reported to be associated with lung cancer. c-Met is a receptor tyrosine kinase ^28-30 , which activates tyrosine kinase activity when bound to the ligand hepatocyte growth factor (HGF), via which angiogenesis, cell motility, and proliferation ^32,33 have been reported to be involved in invasion and differentiation. In this study, the 1234th tyrosine whose phosphorylation was enhanced in response to EMT induction is located in the activation loop (A loop) of c-Met, and is autophosphorylated together with the 1235th tyrosine. -Activate Met. Excessive phosphorylation at the 1234th and / or 1235th tyrosine can lead to cancer growth and invasion, ^34,35 and in lung adenocarcinoma, phosphorylation at the 1235th tyrosine ²⁹ oxide c-Met-overexpressing group has also been reported that they tend lower survival rate compared to the low expression group. These research results are consistent with the results obtained by this study.
As described above, each protein is deeply related to the malignancy of cancer, and the 516th tyrosine of TrkC, 1404th tyrosine of TNS1, the 1234th and / or 1235th of c-Met found in this study A method of using phosphorylation of at least one site of tyrosine as a diagnostic marker is considered useful as a malignancy test method for early lung adenocarcinoma.

Reference paper
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  The patient information of the tissue sample used in Example 1 is shown in the following table.

  The measurement conditions of Example 1 for quantitative analysis by multiple reaction monitoring (MRM) analysis of each peptide are shown in the following table.

[Example 2]
TGF-β treated materials and methods Cell culture
A549 cells (human lung adenocarcinoma cell line) were fixed at 37 ° C using RPMI-1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% (w / v) fetal bovine serum (FBS). Cultivation was performed in a CO ₂ incubator. TGF-β treatment involves culturing cells in low serum (0.1% FBS) medium for 24 hours, followed by 48 hours in medium supplemented with TGF-β (HumanZyme, Chicago, IL, USA, final concentration 5 ng / mL) This was done by culturing. On the other hand, TGF-β non-treatment was performed by culturing for 48 hours in a medium to which only a solvent (DMSO) in which TGF-β was dissolved was added.

Tissue specimens Cancer tissues removed from 58 early stage (Ia or Ib) lung adenocarcinoma patients who agreed to use tissue specimens for research, etc., and frozen tissue specimens stored at -80 ° C until used in this study They were obtained from Kanagawa Cancer Center and Yokohama City University Hospital (Table 3). The breakdown is 22 patients with poor prognosis who relapsed within 5 years after excision and 36 patients with good prognosis who did not relapse within 5 years after surgery.

Protein Preparation Frozen tissue was prepared in 50 mM ammonium bicarbonate solution containing 8 M urea, 4% (w / v) sodium deoxycholate, Protease Inhibitor Mix and Phosphatase Inhibitor Cocktail 2 and 3 using the Sample Grinding Kit. It was crushed. The crushed material was sonicated 5 times (1 second interval) with UR-21P (TOMY, Tokyo, Japan), then centrifuged at 15,000g (4 ° C) for 15 minutes to recover the supernatant, and this was extracted with protein Liquid. The dephosphorylation treatment was performed by reacting 10 unit alkaline phosphatase at 37 ° C. for 1 hour to 50 μg of protein.

Immunoblot analysis Protein extracts were separated using SuperSep ^™ Ace (7.5% T, 17 wells) and transferred to PVDF membrane using the Trans-Blot® Turbo ^™ Transfer System. Then, PVDF membrane was added to T-PBS containing 5% (w / v) skim milk (final concentration 10 mM Na ₂ HPO ₄ · 12H ₂ O, 1.8 mM KH ₂ PO ₄ , 0.137 M NaCl, 2.7 mM KCl, 0.05% [ v / v] Tween20) was allowed to react for 1 hour to perform a blocking operation to suppress nonspecific reactions. As for primary antibodies, rabbit anti-SSFA2 antibody (Proteintech, Chicago, IL, USA), rabbit anti-phosphorylated SSFA2 [Ser92] antibody (self-made scrum request), antibody rabbit anti-actin β antibody 5% (w / v) skim milk The solution diluted 1,000 times with T-PBS was used and allowed to react for 12 hours or more. After the reaction, the PVDF membrane was washed with T-PBS, and a peroxidase (HRP) -labeled goat anti-rabbit IgG antibody was diluted 5,000 times with T-PBS containing 5% (w / v) skim milk as the secondary antibody. And allowed to react for 1 hour. After the reaction, the PVDF membrane was washed with T-PBS, and reacted with HCL substrate ECL Plus Western Blotting Detection Reagents (GE Healthcare) for 5 minutes. The signal of each protein was detected using LAS4000mini luminescence image analyzer (GE Healthcare).

Preparation of peptide solution DTT was added to the protein extract so as to have a final concentration of 10 mM, followed by reduction at room temperature for 45 minutes. After the reduction, iodoacetamide was added to a final concentration of 5 mM, and the reaction was carried out at room temperature for 30 minutes to alkylate cysteine residues. Thereafter, the mixture was centrifuged at 10,000 g for 15 minutes to recover the supernatant, and 3 times the amount of 50 mM ammonium bicarbonate was added to dilute the final concentration of urea to 2 M. After protein quantification using NanoDrop 2000c, trypsin was added to a protein equivalent to 100 μg at a protein: trypsin (enzyme) ratio of 20: 1 (w / w) for 16 hours 37 Incubated at 0 ° C. After adding 20% (v / v) TFA of 1/20 of the digest, centrifuge for 5 minutes at 15,000 g (4 ° C), collect the supernatant, and add peptide by phase transfer dissolution method (PTS method) The sodium deoxycholate contained in the solution was removed (quoted) ¹ . The collected peptide solution was once dried and then redissolved in 0.1% trifluoroacetic acid, and then the peptide was concentrated and desalted using C18 Stage Tip ² . Subsequent phosphopeptide enrichment was performed using Titansphere TiO2 bulk beads (GL Sciences, Tokyo, Japan) ³ . The concentrated phosphorylated peptide was desalted and concentrated again using C18 Stage Tip, and each peptide was stored in a dried state at −20 ° C. until mass spectrometry.

Peptide quantification by multiple reaction monitoring (MRM) analysis Multiple reaction monitoring (MRM) analysis was performed using TripleTOF5600 system connected with cHiPLC nanoflex system (AB Sciex). The separation column connected to the cHiPLC nanoflex system was ChromXP C18-CL, 75 μm id × 15 cm, and the flow rate was 300 nL / min. A buffer contains 0.1% (v / v) formic acid solution containing 2% (v / v) acetonitrile, and B buffer contains 0.1% (v / v) formic acid solution containing 80% (v / v) acetonitrile. Was used. Peptide adsorption is performed with A buffer 98% (v / v) and B buffer 2% (v / v), and elution is continuously increased from 2% to 40% over 42 minutes. Made using the program. Multiple reaction monitoring (MRM) scans are performed in positive mode with ion spray voltage 2300V, curtain gas and spray gas mode set to 20, resolution set to unit, measured mass (m / z) range from 100 to 1600 Da MS2 accumulation times was set to 0.1 seconds. The synthesis of phosphorylated peptides and stable isotope-labeled phosphorylated peptides was performed with scram (Table 4). MRMPilot v2.0 software was used to automatically calculate the precursor peptide / fragment ion pair (MRM transition) and collision energy of the target peptide. Finally, reproducible y ions and MRM transitions were selected (Table 4). For each peptide sample, a stable isotope labeled-phosphorylated peptide at a concentration of 25 fmol was added and used as an internal standard for quantification. The calibration curve was prepared using peptides of 1, 5, 10, and 50 fmol, and the accuracy of the calibration curve was kept within 20% (% CV <20). Mass spectrometry data obtained as a result of mass spectrometry was analyzed using MultiQuant v.2.1, and the amount of peptide was calculated.

Statistical analysis
Statistical analysis such as Mann-Whitney test to show statistical significance of differences between two groups, Kaplan-Meier method to perform recurrence-free survival analysis, receiver operating characteristic (ROC) curve analysis, etc. GraphPad prism 5 (V. 5.04, GraphPad Software, San Diego, CA, USA).

Results From the comprehensive relative quantitative analysis of phosphorylated peptides using a mass spectrometer, the 92nd serine of SSFA2 is phosphorylated as a phosphorylated peptide whose expression level is markedly increased in response to TGF-β treatment in A549 cells. Was found. As a result of immunoblot analysis using an antibody specific to SSFA2 and an antibody specific to SSFA2 phosphorylated at the 92nd serine, the expression level of these proteins in cells does not vary with TGF-β treatment. It was confirmed that phosphorylation at the 92nd serine was enhanced (FIG. 8).
Next, using cryopreserved lung adenocarcinoma tissue of 58 patients removed by surgery, 22 patients who had relapsed lung adenocarcinoma 5 years later (poor prognosis group, PP) and 36 patients who had no recurrence Using the name (good prognosis group, GP) of cancer tissue, immunoblot analysis using each specific antibody and multiple reaction monitoring (MRM) analysis using a mass spectrometer were performed. First of all, SSFA2 content in tissues when tissue samples were divided into cancerous and non-cancerous parts was extracted by immunoblot analysis using SSFA2-specific antibody. Both patients with poor prognosis and good prognosis It was confirmed that SSFA2 was expressed only in the cancerous part of the group (FIG. 9A). Furthermore, as a result of examining the amount of protein in which the 92nd serine of SSFA2 was phosphorylated by immunoblotting analysis using a phosphorylation site-specific antibody, it was found in the cancer part of the poor prognosis group compared with the cancer part of the good prognosis group. It was found that the expression level increased (FIG. 9B).
Furthermore, as a result of investigating the amount of peptide in which the 92nd serine of SSFA2 was phosphorylated in the cancer part of the good prognosis group and poor prognosis group by multiple reaction monitoring (MRM) analysis, it was found that the Significant difference (p <0.005) was observed (FIG. 10). Based on the obtained results, a relapse-free survival curve for each phosphopeptide level was prepared. As a result, multiple response monitoring (MRM) was performed for 20 cases with poor prognosis in stage I lung adenocarcinoma and 31 cases with good prognosis. ) When the amount of phosphopeptide obtained by analysis is classified as above average or below, the group above average value has a statistically significantly shorter postoperative survival time than the group below average value (p <0.005). (Fig. 11). Moreover, the result of the receiver operating characteristic (ROC) curve analysis showed that the protein in which the 92nd serine of SSFA2 was phosphorylated was useful as a diagnostic marker (FIG. 12).

Consideration
SSFA2 has been identified as a gene whose expression is increased by activated K-ras in colon cancer cell lines and is also called K-ras-induced actin-interacting protein (KRAP) ⁴ . The function of SSFA2 is to regulate the release of calcium ions by binding to inositol triphosphate receptor (IP3R) present on the endoplasmic reticulum and nuclear membrane, and to regulate various physiological actions in the cell. There are reports of ⁵ . Furthermore, SSFA2 is involved in the accumulation of IP3R in the vicinity of tight junctions (cell-cell adhesion structures) during differentiation of MDCK cells (cell line derived from canine kidney tubular epithelial cells) together with vimentin, including epithelial cells It has been suggested to regulate the localization of IP3R in various cell types ⁶ . Also, the operation of the IP3R is ⁷ has associated with EMT is suggested, EMT induction in breast cancer cells has also been reported to be dependent on calcium signaling ^8. These reports suggest that SSFA2 is likely related to EMT induction. On the other hand, the role of SSFA2 phosphorylation has not yet been elucidated. This is the first report that phosphorylation of SSFA2 at the 92nd serine is closely related to EMT induction and prognosis of early lung adenocarcinoma. Based on the above results, it is considered that detecting the phosphorylation of the 92nd serine of SSFA2 is useful as a method for examining the malignancy of early lung adenocarcinoma.
Furthermore, SSFA2 in colon cancer cell lines and lung squamous carcinoma tissues, ⁴ has been identified as a tumor-specific ^{genes, 9.} In this study as well, the results of immunoblot analysis confirmed that SSFA2 is a protein specifically expressed in the cancerous part and can be used as a lung adenocarcinoma detection marker. This also has the potential to be used as a therapeutic target that acts specifically only in the cancerous part without acting on normal tissues.

Reference paper
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  The patient information of the tissue specimen used in Example 2 is shown in the following table.

  The measurement conditions of Example 2 for quantitative analysis by multiple reaction monitoring (MRM) analysis of each peptide are shown in the following table.

  The present invention can be used as an early screening test for predicting prognosis of patients with early lung adenocarcinoma.

<SEQ ID NOS: 1-16>
SEQ ID NOs: 1 to 16 show the amino acid sequences of internal standard peptides used in the multiple reaction monitoring (MRM) analysis of Examples 1 and 2.
Phospho-TNS1 (pY1404): AGSLPNY [Pho] ATINGK (SEQ ID NO: 1)
Isotope-labeled phospho-TNS1 (pY1404): AGSLPNY [Pho] ATINGK ( ¹³ C ₆ , ¹⁵ N ₂ ) (SEQ ID NO: 2)
Phospho-c-Met (pY1234): DMYDKEY [Pho] YSVHNK (SEQ ID NO: 3)
Isotope-labeled phospho-c-Met (pY1234): DMYDKEY [Pho] YSVHNK ( ¹³ C ₆ , ¹⁵ N ₂ ) (SEQ ID NO: 4)
Phospho-TrkC (pY516): IPVIENPQY [Pho] FR (SEQ ID NO: 5)
Isotope-labeled phospho-TrkC (pY516): IPVIENPQY [Pho] FR ( ¹³ C ₆ , ¹⁵ N ₄ ) (SEQ ID NO: 6)
Phospho-SSFA2 (pS92): TPLGAS [Pho] LDEQSSSTLK (SEQ ID NO: 7)
Isotope-labeled Phospho-SSFA2 (pS92): TPLGAS [Pho] LDEQSSSTLK ( ¹³ C ₆ , ¹⁵ N ₂ ) (SEQ ID NO: 8)
TNS1 (Y1404): AGSLPNYATINGK (SEQ ID NO: 9)
Isotope-labeled TNS1 (Y1404) ： AGSLPNYATINGK ( ¹³ C ₆ , ¹⁵ N ₂ ) (SEQ ID NO: 10)
c-Met (Y1234): DMYDKEYYSVHNK (SEQ ID NO: 11)
Isotope-labeled c-Met (Y1234): DMYDKEYYSVHNK ( ¹³ C ₆ , ¹⁵ N ₂ ) (SEQ ID NO: 12)
TrkC (Y516): IPVIENPQYFR (SEQ ID NO: 13)
Isotope-labeled TrkC (Y516): IPVIENPQYFR ( ¹³ C ₆ , ¹⁵ N ₂ ) (SEQ ID NO: 14)
SSFA2 (S92): TPLGASLDEQSSSTLK (SEQ ID NO: 15)
Isotope-labeled SSFA2 (S92): TPLGASLDEQSSSTLK ( ¹³ C ₆ , ¹⁵ N ₂ ) (SEQ ID NO: 16)
<SEQ ID NO: 17>
SEQ ID NO: 17 is the amino acid sequence of TPLGA (pS) LDEQS. Cys added at the N-terminus to bind to carrier protein (C + TPLGA (pS) LDEQS) was used as an immunogen for antibody production (antibody against SSFA2 in which the 92nd serine of SSFA2 was phosphorylated) .

Claims (14)

At least one selected from the group consisting of TrkC 516 tyrosine, TNS1 1404 tyrosine, SSFA2 92 serine for examining the malignancy of early lung adenocarcinoma in a subject-derived specimen A method to detect phosphorylation at one site. Early lung adenocarcinoma is metastasized and / or has an increased phosphorylation of at least one site selected from the group consisting of TrkC at 516 tyrosine, TNS1 at 1404 tyrosine, and SSFA2 at 92nd serine. Or the method of Claim 1 which shows that possibility of recurrence is high. The method according to claim 1 or 2, further comprising detecting phosphorylation of one or both tyrosine at 1234/1235 of c-Met. 4. The method according to claim 3, wherein phosphorylation of one or both tyrosine at 1234/1235 of c-Met is increased, indicating that there is a high possibility that early lung adenocarcinoma metastasizes and / or recurs. The method according to claim 1, wherein the subject-derived specimen is lung adenocarcinoma tissue, serum, whole blood, or urine. TrkC antibody against TrkC phosphorylated at 516th tyrosine, TNS1 antibody against TNS1 phosphorylated at 1404th tyrosine, SSFA2 antibody against SSFA2 phosphorylated at serine 92th The method according to any one of claims 1 to 5, wherein the expression level of the phosphorylated protein is measured by an antigen-antibody reaction using at least one antibody. Furthermore, an antigen-antibody reaction using an antibody against c-Met in which the 1234th tyrosine of c-Met was phosphorylated and / or an antibody against c-Met in which the 1235th tyrosine of c-Met was phosphorylated was used to produce phosphorylation. The method according to claim 6, wherein the expression level of oxidized c-Met is measured. Multiple reaction monitoring (MRM) analysis detects phosphorylation of at least one site selected from the group consisting of 516th tyrosine of TrkC, 1404th tyrosine of TNS1 and 92th serine of SSFA2. 6. The method according to any one of 5. Furthermore, the method of Claim 8 which detects phosphorylation of the 1234 / 1235th tyrosine of c-Met by multiple reaction monitoring (MRM) analysis. The method of Claim 9 which performs a multiple reaction monitoring (MRM) analysis on the conditions shown in the following table | surface.
The method according to claim 1, wherein the examination is performed before and / or after the operation. At least one selected from the group consisting of a reagent capable of detecting phosphorylation of the 516th tyrosine of TrkC, a reagent capable of detecting phosphorylation of the 1404th tyrosine of TNS1, and a reagent capable of detecting the phosphorylation of the 92nd serine of SSFA2. Early lung adenocarcinoma test kit containing two reagents. The kit according to claim 12 , further comprising a reagent capable of detecting phosphorylation of 1234th and / or 1235th tyrosine of c-Met. The kit according to claim 12 or 13, wherein the reagent is an antibody.