JP6537095B2 - Markers for predictive diagnosis of cancer recurrence and / or metastasis - Google Patents

Description

  The present invention relates to a marker for predictive diagnosis of cancer prognosis, specifically, a marker for predictive diagnosis of cancer recurrence and / or metastasis.

The p62 protein has been elucidated to collect damaged proteins in cells to perform autophagy, and it is known that the p62 protein accumulates in cancer tissues (Non-patent Documents 1 to 5).
Heretofore, in order to investigate the relapsing and metastatic properties of cancer, it has usually been possible only to collect the cancer itself (biopsy). However, the addition of a biopsy into the tissue was considered to be undesirable, as it could cause the tumor to metastasize.

Cell, 2007; 131 (6): 1149-1163 Cell, 2009; 137 (6): 1062-1075 J. Cell Biol., 2011; 193 (2): 275-284. Cancer Sci., 2012; 103 (4): 760-766 Onco Targets Ther., 2013; 6: 883-888

  Under such circumstances, it has been desired to develop a method capable of predicting and diagnosing recurrence and metastasis of cancer without using cancer tissue itself.

  The present invention has been made in consideration of the above situation, and includes the following markers for predictive diagnosis of cancer recurrence and / or metastasis, compositions for predictive diagnosis of cancer recurrence and / or metastasis, cancer recurrence and The present invention provides a kit for predicting diagnosis of metastasis, a method for detecting a condition in which recurrence and / or metastasis of cancer are predicted, and the like.

(1) A marker for predicting cancer recurrence and / or metastasis, which comprises p62 protein.
(2) A composition for predicting and diagnosing recurrence and / or metastasis of cancer, which comprises a substance capable of detecting p62 protein.
In the composition of the above (2), the substance capable of detecting the p62 protein includes, for example, a substance capable of staining the p62 protein or an antibody against the p62 protein.

(3) A kit for prognostic diagnosis of cancer recurrence and / or metastasis, comprising a substance capable of detecting p62 protein.
In the kit of (3) above, examples of the substance capable of detecting the p62 protein include a substance capable of staining the p62 protein, an antibody against the p62 protein, and the like.

(4) A substance capable of detecting the p62 protein is made to react with a biological sample derived from a cancer tissue or a normal tissue around the precancerous tissue to detect the p62 protein, and the obtained result is used as an index for recurrence and / or metastasis of cancer. How to detect the state that is predicted.
In the method (4) above, examples of the substance capable of detecting the p62 protein include a substance capable of staining the p62 protein, an antibody against the p62 protein, and the like.

  According to the present invention, it is possible to predict future metastasis and recurrence of cancer without cutting into cancer tissue, and at the same time, it is possible to predict cancer tissue and precancerous tissue lesions regardless of cancer specific substances because of the nature of p62 protein. From the viewpoint of abnormality in the phenomenon of autophagy (over-accumulation of p62 protein) in normal tissues of cancer, predictive markers for predictive diagnosis capable of predictive diagnosis of metastasis and recurrence of cancer, and predictive diagnosis of recurrence and / or metastasis of cancer It is possible to provide a composition for use as well as a method for detecting a state in which cancer recurrence and / or metastasis are predicted.

  According to the present invention, functionally normal (ie, p62 protein in the above normal tissue) even in the absence of morphological abnormal findings in normal tissue around cancerous tissue or precancerous tissue lesions. Abnormal expression), recurrence of cancer after cancer treatment and possibility of metastasis (prognosis is good or bad) can be predicted. Therefore, in the follow-up observation, it is possible to focus more on the presence or absence of onset of recurrent carcinogenesis or metastatic cancer, and if onset is possible, treatment can be promptly carried out. The invention is very useful.

It is a figure which shows the correlation of the expression level of p62 protein, p53 protein, and ki67 protein in the specimen which carried out the surgery alone and the specimen which combined chemoradiotherapy. It is a figure which shows the expression state of p62 and p53. It is a figure which shows the correlation with the expression state of p62 and the recurrence and metastasis in the normal epithelium around a tumor part.

  Hereinafter, the present invention will be described in detail. The scope of the present invention is not restricted by these explanations, and it can be suitably changed and implemented in the range which does not spoil the meaning of the present invention also except the following examples. All publications cited in this specification, such as prior art documents, and published publications, patent publications and other patent documents, are incorporated herein by reference.

1. SUMMARY OF THE INVENTION Head and neck cancer is an area exposed to oxidative stress such as tobacco and alcohol. Therefore, it is important to protect the cells from oxidative stress, the bioprotective mechanism transcription factor NF-E2-related Factor 2 (Nrf2) is important, and a protein called Keap1 acts as an intracellular redox sensor and regulates Nrf2 activity. I know.

  When the p62 protein (hereinafter, also simply referred to as "p62") accumulates in cells excessively, it binds to keap1 and induces gene expression of antioxidant proteins. In cancer cells, p62 is accumulated excessively (autophagy), and it has a survival strategy to protect itself from oxidative stress (Cell, 2007; 131 (6): 1149-1163). This excessive accumulation of p62 has been reported in hepatocellular carcinoma, prostate cancer, etc., and has not been reported in head and neck cancer, but recently, in oral squamous cell carcinoma, if p62 is excessively accumulated in cells, It has been reported that it is resistant to radiation therapy and has a poor prognosis (Inui et al, PlosOne, 2013). Therefore, the inventor of the present application performed p62 immunostaining on a surgical specimen (68 cases) of oral squamous cell carcinoma at the University of Tsukuba dental and oral surgery.

  As a result, it is recognized that the expression of p62 is high as cancer turns from normal epithelium, and in particular, cases where p62 is expressed in the normal epithelium around the tumor part or precancerous part are cases of cancer recurrence and metastasis. There was a surprising event that there was a significant increase in (especially in oral cancer patients, the risk of metastasis / relapse is 8 times higher when p62 is expressed in normal tissues). From this, the present inventor has found that predictive diagnosis of metastasis and recurrence of cancer is possible from the viewpoint of autophagy abnormalities, and completed the present invention.

2. Markers for Predictive Diagnosis of Recurrence and / or Metastasis of Cancer The markers for predictive diagnosis of recurrence and / or metastasis of cancer of the present invention (hereinafter, also referred to as "the markers for predictive diagnosis of the present invention") are p62 as described above. Is included. When expression of p62 is found in a biological sample of a test animal, in particular, a biological sample derived from a normal tissue around a cancer tissue or a precancerous tissue, the expression of p62 is not more than that in the case of no such expression. It can be predicted and judged that cancer is likely to relapse and / or metastasize (the risk of recurrence and / or metastasis is high).

In the present invention, cancers (cancer types) targeted for predictive diagnosis of recurrence and / or metastasis are not particularly limited, and all cancer types can be targeted for predictive diagnosis, for example, oral cancer, pharynx Specific examples include cancer, esophagus cancer, lung cancer, cervical cancer, etc. Among them, in the case of oral cancer, the possibility of recurrence and / or metastasis can be predicted (with higher accuracy) more highly predictively. preferable.
In the present invention, cancer tissue refers to a malignant tumor derived from epithelial tissue, and precancerous tissue refers to a morphologically altered tissue that is more likely to develop cancer than normal tissue. Also, normal tissue around these cancer tissues and precancerous tissue means non-cancerous tissue and non-precancerous tissue to be removed by providing a safety margin at the time of surgical removal of cancer, for example, normal Epithelial tissue, normal glandular tissue and the like.

3. Composition for Predictive Diagnosis of Recurrence and / or Metastasis of Cancer The composition for prediction and diagnosis of recurrence and / or metastasis of cancer of the present invention (hereinafter, also referred to as "the composition for predictive diagnosis of the present invention") is the aforementioned. As a result, it is a composition (pharmaceutical composition) characterized by containing a substance capable of detecting p62.
The present invention is also directed to a method for predicting and diagnosing a cancer recurrence and / or metastasis, which comprises using a substance capable of detecting p62, and for producing a drug for predicting and diagnosing a cancer recurrence and / or metastasis. It includes the use of a substance capable of detecting the p62 protein, and the use of a substance capable of detecting the p62 for making a predictive diagnosis of recurrence and / or metastasis of cancer.

(1) Substances that can detect p62
Examples of substances capable of detecting p62 include, for example, substances capable of staining p62, antibodies against p62, and the like, and combinations thereof may also be used.
As the substance capable of staining p62, p62 present in a biological sample derived from a normal tissue from cancer tissue or precancerous tissue can be detected using a known detection means / detection method (eg, immunohistochemical staining, western blotting, etc.) The substance is not particularly limited as long as it is a substance that can be detected, and examples include an antibody and the like. Substances capable of staining p62 can also be used in staining by immunostaining (antibody staining) together with an antibody against p62 described later.

Further, an antibody against p62 (anti-p62 antibody) can be prepared as follows.
(A) Preparation of polyclonal antibody
(i) Preparation of Antigen and its Solution In preparation of anti-p62 antibody, first, it is necessary to prepare or obtain a protein to be used as an immunogen (antigen).
The antigen protein may be, but is not limited to, purified p62, and is not limited to this. For example, it consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of p62 It is also possible to use a protein having an activity equivalent to As p62, for example, mammalian-derived p62, specifically, human-derived p62, mouse-derived p62, rat-derived p62 or the like can be used. Here, the nucleotide sequence and amino acid sequence information of human and mouse-derived p62 can be obtained from the following accession numbers, respectively.

<Human-derived p62>
GenBank accession number: M88108.1 (base sequence; SEQ ID NO: 1), AAA 59990.1 (amino acid sequence; SEQ ID NO: 2)
<Mouse-derived p62>
GenBank accession number: U17961.1 (base sequence; SEQ ID NO: 3), AAA86693.1 (amino acid sequence; SEQ ID NO: 4)

  The method for preparing purified p62 to be an antigen is not limited, but, for example, p62 is produced using a transformant of a suitable host cell (cultured fibroblasts of human origin, Chinese hamster ovary cells, yeast, etc.) And common biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., which may be used alone or in combination. .

  Next, the purified p62 obtained is dissolved in a buffer to prepare an antigen solution. At that time, if necessary, an adjuvant may be added to effectively carry out the immunization. As the adjuvant, for example, commercially available Freund's complete adjuvant or Freund's incomplete adjuvant can be used. These adjuvants may be used alone or in combination of two or more, and are not limited.

(ii) Immunization and Collection of Antisera Immunization is performed by administering a solution containing the above purified p62 to a mammal (eg, mouse, rat, rabbit, etc.). Administration is mainly performed by intravenous, subcutaneous or intraperitoneal injection.
The dose per dose of the antigen solution is not limited, and is preferably, for example, an amount such that the purified p62 is 2 to 500 μg per animal, more preferably 10 to 100 μg.
The administration interval is not limited, and is preferably, for example, several days to several weeks, and more preferably two to three weeks. Moreover, the frequency | count of administration is 2 to 10 times, for example.

Although collection of the serum (antisera) obtained by the above-mentioned immunization is not limited, for example, it is preferable to carry out after 1 to 28 days from the day of the final administration, and more preferably after 2 to 14 days. The antisera can be collected from the blood of the immunized animal according to a conventional method.
(iii) Selection of target antisera From the antisera collected from each of the immunized animals, a target antisera, ie, an antisera containing an anti-p62 antibody, is screened.

  The screening method is not limited. For example, the collected antisera and purified p62 as the antigen or recombinant p62 (produced by human-derived cultured fibroblasts, Chinese hamster ovary cells, yeast, etc.) or those A known immunoassay (immunoassay) can be performed according to a conventional method using a mutant enzyme of Immunoassays include labeled immunoassays and turbidimetric immunoassay (TIA). Among them, the former is preferred. For example, in addition to enzyme immunoassays (EIA) such as ELISA, radioimmunoassay (RIA) And fluorescence immunoassay (FIA) etc. are preferably mentioned. Moreover, as a labeled immunoassay, a Western blot method (combination with electrophoresis) etc. is preferably mentioned as what used combination with other separation methods. In the case of Western blotting, a protein sample to be detected is used after being denatured by heat and a surfactant, so that the antigen in the screening method is a mutant enzyme.

The anti-p62 antibody contained in the target antiserum by the above screening is usually a polyclonal antibody. When it is necessary to purify the antibody, for example, known purification methods such as ammonium sulfate precipitation, ion exchange chromatography, affinity chromatography, gel chromatography, etc. may be used appropriately in combination of only one or two or more. You can do it.
The reactive site in p62 to which the anti-p62 antibody can specifically bind (recognize) is not particularly limited.

(B) Production of monoclonal antibody
(i) Preparation of Antigen and Its Solution Preparation of an antigen and its solution can be carried out in the same manner as in the production of the above-mentioned polyclonal antibody.
(ii) Immunization and Collection of Antibody-Producing Cells The same method and conditions as in the preparation of the above-mentioned polyclonal antibody can be adopted for the immunization method, the dose using the above antigen solution, the administration interval and the administration frequency.

Although collection of cells (antibody producing cells) producing anti-p62 antibody obtained by the above immunization is not limited, for example, it is preferable to carry out after 1 to 14 days from the day of final administration, more preferably after 2 to 4 days It is.
Preferred examples of antibody-producing cells include spleen cells, lymph node cells (particularly, regional lymph node cells), peripheral blood cells, etc. Among them, spleen cells and regional lymph node cells (eg, knee lower lymph node cells) Is more preferred.

(iii) Cell fusion A fused cell (hybridoma) can be obtained by performing cell fusion between the collected antibody-producing cells and myeloma cells.
As the myeloma cells, for example, cell lines generally available in mammals such as mice can be used. Specifically, a cell line having drug selectivity, which can not grow in a HAT selection medium (a medium containing hypoxanthine, aminopterin and thymine) in an unfused state but can grow in a fused state with antibody-producing cells preferable. Specifically, for example, PAI, P3X63-Ag.8.U1 (P3U1), NS-I or the like can be used as mouse myeloma cells.

The above cell fusion is performed, for example, by mixing antibody-producing cells and myeloma cells in a culture medium for animal cell culture such as RPMI-1640 medium without serum and causing a fusion reaction. The mixing ratio of antibody-producing cells to myeloma cells is, for example, 5: 1.
In general, the fusion reaction is preferably performed in the presence of a cell fusion promoter, and polyethylene glycol having an average molecular weight of 1000 to 6000 daltons can be used as the promoter. Alternatively, antibody-producing cells can be fused with myeloma cells using a commercially available cell fusion device utilizing electrical stimulation (eg, electroporation).

The cells after the fusion reaction are cultured, for example, in HAT selective medium. After the above culture, cells in which growth in HAT selection medium is observed become fused cells (hybridomas).
(iv) Selection of Target Hybridoma and Cloning Thereof From the hybridoma obtained by the above culture, a target hybridoma, ie, a hybridoma that produces an anti-p62 antibody is screened. Specifically, the culture supernatant is screened for the presence of the target anti-p62 antibody.

  Although the screening method is not limited, for example, a part of the culture supernatant is collected and purified as antigen p62 or recombinant p62 (cultured fibroblasts derived from human, Chinese hamster ovary cells, yeast etc. ) Or their mutant enzymes, known immunoassays (immunoassay) can be carried out using their usual methods. As the immunoassay, for example, in addition to enzyme immunoassay (EIA) such as ELISA, radiation immunoassay (RIA), fluorescence immunoassay (FIA) and the like are preferably mentioned. In addition, Western blotting (combination with electrophoresis) and the like are preferably mentioned. In the case of Western blotting, a protein sample to be detected is used after being denatured by heat and a surfactant, so that the antigen in the screening method is a mutant enzyme.

The anti-p62 antibody contained in the culture supernatant of the target hybridoma after the above screening is an antibody obtained before cloning of the hybridoma, but may be an antibody consisting of a single molecule (monoclonal antibody), There is no limitation.
The reactive site in p62 to which the anti-p62 antibody can specifically bind (recognize) is not particularly limited.

Cloning of a target hybridoma by the above screening, that is, establishment of a monoclonal antibody-producing cell line can be generally performed by selecting colonies derived from one cell by culture using a limiting dilution method or the like.
(v) Collection of Monoclonal Antibody The method for collecting a monoclonal antibody from the hybridoma obtained by the above cloning is not limited, but in general, a cell culture method or an ascites fluid formation method can be adopted.

In the cell culture method, hybridomas obtained by cloning are cultured, for example, in an animal cell culture medium under conditions of 37 ° C., 5% CO ₂ for 7 to 14 days, and the target culture supernatant is obtained from the culture supernatant thereafter. Monoclonal antibodies can be collected.
In the ascites fluid formation method, for example, about 10 ⁶ hybridomas obtained by cloning are administered per animal into the peritoneal cavity of a mammal of the same lineage as the mammal from which the myeloma cells used in cell fusion are derived. Then, the hybridoma can be grown in large scale, and the target monoclonal antibody can be collected from ascites fluid or serum collected 7 to 14 days after administration.

  In either the cell culture method or the ascites formation method, when purification of the antibody is required during or after collection of the monoclonal antibody, ammonium sulfate precipitation, ion exchange chromatography, affinity chromatography, gel chromatography, etc. Known purification methods can be appropriately adopted and carried out using only one or a combination of two or more.

(2) Compounding ratio of substance capable of detecting p62 protein In the composition for prediction diagnosis of the present invention, compounding of a substance capable of detecting p62 protein (a substance capable of staining p62, an anti-p62 antibody, or a combination thereof) The proportion is not particularly limited, but preferably 1 to 100% by weight, more preferably 10 to 100% by weight.

(3) Other components The composition for predictive diagnosis of the present invention may contain other components in addition to the substance capable of detecting the p62 protein, as long as the effects of the present invention are not significantly impaired. There is no limitation. For example, a primary antibody detection reagent, a chromogenic substrate and the like can be included.

(4) Detection of p62
The detection of p62 can be carried out by detecting p62 by reacting a substance capable of detecting p62 protein with a biological sample derived from a cancer tissue or a normal tissue around a precancerous tissue.
As a specific p62 detection method, known detection methods such as, for example, an immunoassay, an immunostaining method and the like can be used according to the usual method.

  For example, as the immunoassay, there are labeled immunoassay (including immunostaining) and immunoturbidimetric assay (TIA), and among them, the former is preferable. For example, enzyme immunoassay such as ELISA ( Besides EIA), radiation immunoassay (RIA), fluorescence immunoassay (FIA) and the like are preferably mentioned. Moreover, as a labeled immunoassay, a Western blot method (combination with electrophoresis) etc. is preferably mentioned as what used combination with other separation methods. Among these, ELISA and / or Western blotting are preferred. Alternatively, detection may be performed using one or more anti-p62 monoclonal antibodies and combining two or more immunoassays.

(5) Detection (evaluation) of a condition that predicts recurrence and / or metastasis of cancer
In the present invention, based on the detection result of p62 detected as described above, a state in which cancer recurrence and / or metastasis are predicted can be detected (assessed).
That is, according to the present invention, a substance capable of staining p62 and a biological sample derived from a normal tissue from a cancer tissue or a precancerous tissue are reacted to detect p62 protein, and the obtained result is used as an index for recurrence and / or Or including methods of detecting conditions in which metastasis is predicted.

  Here, "detecting a condition in which cancer recurrence and / or metastasis is predicted" means that the possibility of recurrent cancer or metastatic cancer is significantly (prognostically) developed for the cancer that is currently developing. Means to detect the state of. In particular, for oral cancer, when expression of p62 is detected in normal tissues (specifically, normal epithelium), the risk of metastasis / relapse is about eight times higher than when it is not detected. It has been confirmed by the inventor. Therefore, if the expression of p62 is detected in a biological sample derived from normal tissue around cancerous tissue or precancerous tissue, the possibility of metastasis / recurrence is considered even after the currently developing cancer is cured. It can be evaluated that sufficient follow-up is necessary.

4. The present invention relates to the prediction of cancer recurrence and / or metastasis, including the composition for prediction and diagnosis described in the third paragraph (in particular, a substance capable of detecting p62 protein). Includes a diagnostic kit.
When the kit contains a substance capable of detecting p62 protein, the substance is, for example, in the form of powder or solution (pure water, in consideration of its stability (preservability) and ease of use, etc. They may be stored under the conditions of refrigeration or freezing (-30 ° C to -80 ° C) in a state of being dissolved in a buffer, physiological saline or the like). In particular, when the kit contains an anti-p62 antibody, it is preferable that the antibody is prepared in a dissolved state in consideration of stability (conservability) and ease of use.

The kit can include other components in addition to the substance capable of detecting the p62 protein described above. As other components, for example, reagents for primary antibody detection and chromogenic substrates that can be used for ELISA and Western blot can be mentioned.
The kit only needs to have at least a substance capable of detecting the above p62 protein as a component. Therefore, all of the components that are essential for the predictive diagnosis of cancer recurrence and / or metastasis may or may not be provided with the p62 protein together with the detectable substance, and it is not particularly limited. It is not done.

  EXAMPLES The present invention will be more specifically described below with reference to examples, but the present invention is not limited to these.

<Procedure>
We used surgical specimens (68 cases) of patients with squamous cell carcinoma of the oral cavity who visited the University of Tsukuba Dental and Oral Surgery. A 2 μm-thick unstained slide is deparaffinized, antigen-activated in citric acid + microwave, and treated with 0.3% H ₂ O ₂ + methanol Quenching. After blocking, primary antibody treatment with anti-p62 protein antibody (Anti-SQSRM1 / p62 antibody (ab56416); manufactured by Abcam) and secondary antibody treatment (vecta stain kit; manufactured by Vector Labs), avidin-biotin complex The body was treated (vecta stain kit; manufactured by Vector Labs) and developed with DAB (manufactured by Dako). After staining with hematoxylene, it was mounted.

<Result>
Among the surgical specimens (68 cases), the expression levels of p62 protein, p53 protein, and ki67 protein were significantly different between the surgical alone samples (45 cases) and the specimens (23 cases) combined with chemoradiotherapy. There was no difference (Figure 1).
While p62 expression is increased from normal epithelium to the area of cancer, in dysmorphic epithelia, p53 protein was frequently stained in cells invading cancer from the basal cell layer, but p62 protein was Many staining was observed in the spiny cell layer. The positive examples in heterotypic epithelium were 18/68 (26.4%) of p53 protein and 40/68 (58.8%) of p62 protein (FIG. 2).

From normal epithelium to the area of cancer, p62 expression was also higher and correlated with p53 expression.
Cases in which p62 protein was expressed in the normal epithelium around the tumor site were significantly more frequently associated with recurrent cancer and metastatic cancer (FIG. 3). From this, it was found that it is possible to predict metastasis and recurrence of cancer from the viewpoint of abnormality of the phenomenon of autophagy (excess accumulation of p62 protein).

Claims (7)

A marker for predictive diagnosis of cancer recurrence and / or metastasis, characterized in that it comprises p62 protein in normal tissue around cancerous tissue or precancerous tissue . A composition for prognostic diagnosis of cancer recurrence and / or metastasis, comprising a substance capable of detecting p62 protein in normal tissue around cancerous tissue or precancerous tissue . Wherein the detectable substance is, the dyeable material p62 protein, or an antibody against the p62 protein composition of claim 2. A kit for prognostic diagnosis of cancer recurrence and / or metastasis, comprising a substance capable of detecting p62 protein in normal tissue around cancer tissue or precancerous tissue . Wherein the detectable substance is, the p62 protein capable of staining substances, or an antibody against the p62 protein, according to claim 4 kit according. and a detectable substance p62 protein, is reacted with a biological sample of normal tissue from the surrounding cancerous tissue near or precancerous tissue detects the p62 protein, recurrence of cancer results obtained as an indicator and / or metastasis prediction To detect the condition being   The method according to claim 6, wherein the detectable substance is a substance capable of staining p62 protein or an antibody against p62 protein.