JP5942340B2 - How to diagnose cancer malignancy - Google Patents

Description

  The present invention relates to a method for diagnosing malignancy of cancer. More specifically, the present invention relates to a diagnostic method using EMT (Epithelial-mesenchymal transition) as an index in the diagnosis of malignancy of cancer.

  Alternative splicing is one of the important mechanisms for generating protein diversity by generating multiple different transcripts from a single gene. Alternative splicing is strictly controlled in time and space according to the stage of development and organization. This failure of regulation is known to lead to various diseases including cancer, and in recent years, the expression of abnormal splicing variants in cancer cells may be greatly involved in the development and progression of cancer. Has been reported.

  The degree of malignancy of cancer indicates the degree of influence of cancer on a patient who is a host, and is a criterion for selection of a treatment method. Factors that determine the malignancy of cancer include poor prognosis and high metastatic potential, but the malignancy is not uniform even for the same cancer. If the diagnosis of malignancy is appropriately performed, an optimal treatment method suitable for each patient can be performed, and high-quality medical care becomes possible. Specifically, it is possible to examine whether or not to use an anticancer agent after surgery and whether or not to use a highly effective therapeutic agent even if the side effects are strong.

EMT (epithelial-mesenchymal transition) as an indicator of cancer malignancy
The acquisition of is attracting attention. EMT is a phenomenon in which epithelial cells lose their adhesion and cytoskeleton components, which are traits as epithelium, and change morphologically and functionally into mesenchymal cells. EMT was originally a phenomenon observed during development, but other than that, it has acquired EMT-like traits due to high-grade cancer cells, recurrent cancers, and distant metastases. It has become clear.

To date, EMT has transformed cancer cells into poorly differentiated traits to enhance invasion and metastasis, and fibroblasts in the cancer microenvironment, which are also factors that enhance cancer malignancy. It is known that EMT is also involved in the generation of. Therefore, if the acquisition of EMT in cancer cells can be evaluated, it will be possible to diagnose the malignancy of cancer.

A plurality of factors such as Snail family, δEF1 family, and Twist are known as transcription factors that induce EMT. Pathological studies have shown that cancers with higher expression of EMT-inducing transcription factors have higher malignancy and higher expression in cancers that have metastasized or recurred (Non-Patent Documents 1 to 3). .

  However, these transcription factors are expressed differently depending on cancer tissues and cells. Therefore, the EMT inducer is not sufficient to be a common index for EMT acquisition of various cancers. There are other malignant diagnostic indexes specific to specific cancers (Patent Documents 1 to 3), but there is no established malignant diagnostic method that can be commonly used for various cancers.

WO2008 / 096416 JP2010-522884 JP2006-094726

Shirakihara T, Horiguchi K, Miyazawa K, Ehata S, Shibata T, Morita I, Miyazono K, Saitoh M. EMBO J. 2011 Jan 11; 30: 783- 795 Role of Ras signaling in the induction of snail by transforming growth factor-β.Horiguchi K, Shirakihara T, Nakano A, Imamura T, Miyazono K, Saitoh M. J Biol Chem. 2009 Jan 2; 284 (1): 245-53 Differential regulation of epithelial and mesenchymal markers by dEF1 proteins in epithelial mesenchymal transition induced by TGF-β. Shirakihara T, Saitoh M, Miyazono K. Mol. Biol. Cell. 2007 Sep; 18 (9): 3533-4

An object of the present invention is to provide a method for diagnosing malignancy common to various cancers, not limited to tissues and cells, by diagnosing malignancy of cancer using EMT acquisition as an index.

The present inventor has conducted extensive studies to solve the above-mentioned problems, and has identified ESRP (Epithelial Splicing Regulatory Proteins), which is an epithelial-specific splicing regulator, as a target gene common to EMT-induced transcription factors. It was found that ESRP expression was suppressed in high-grade cancer, and the present invention was completed based on this finding.

  That is, the present invention relates to a method for diagnosing the malignancy of cancer by detecting the expression of ESRP in a sample obtained from a patient and using the expression level of ESRP as being suppressed. More specifically, when the EMT-inducible transcription factor increases, the expression level of ESRP as its target gene is suppressed. When ESRP expression is suppressed, induction into EMT is promoted, invasion and acquisition of metastatic potential occur, and cancer malignancy is increased. Therefore, the malignancy of cancer can be diagnosed by using the expression of ESRP as an index.

  Samples obtained from patients include cancerous tissues and tissues suspected of having cancer. Moreover, the value of ESRP in a healthy person is used as a reference value for comparing the expression level of ESRP in a sample obtained from a patient. Or, by using ESRP values in samples at different points in the treatment period, such as samples obtained earlier from patients or samples obtained at the end of treatment, treatment effects, etc. It becomes possible to evaluate about.

ESRP expression may be evaluated by mRNA expression level or protein expression level. The method for evaluating the expression level of mRNA or protein is not particularly limited.
When the target cancer is breast cancer, suppression of ESRP expression and EMT-induced transcription factor δEF-1 and / or
Alternatively, the present invention provides a method for diagnosing the malignancy of cancer by using an increase in SIP1 as an index. The inventor has clarified that EMT-induced transcription factor δEF-1 and / or SIP1 is increased in breast cancer in EMT induction, and diagnoses in combination with suppression of ESRP makes a more accurate diagnosis be able to.

When the target cancer is pancreatic cancer, the present invention provides a method for diagnosing malignancy of cancer using as an index a combination of suppression of ESRP expression and increase in EMT-induced transcription factor δSnail. The inventor has clarified that EMT induction transcription factor Snail is increased in EMT induction in pancreatic cancer, and diagnosis can be performed with higher accuracy by making a diagnosis in combination with suppression of ESRP.

Furthermore, if suppression of ESRP expression can be suppressed, it will lead to suppression of EMT acquisition. Therefore, it can be applied to screening methods for anticancer agents using ESRP expression level as an index. Specifically, anticancer drug candidate substances may be administered to cancer cells or tissues, and then ESRP expression may be detected, resulting in an increase in ESRP expression levels before administration of the candidate substance. If it can be confirmed, it is a method for evaluating that the candidate substance is a substance that can be an anticancer drug.

According to the present invention, ESRP, which is a common target factor for EMT inducers, can be used for diagnosis of cancer malignancy.
Inhibition of the expression of can be used as an indicator. This makes it possible to diagnose the malignancy of cancer that is not limited to tissues and cells, and to select an optimal treatment for each patient. It can also be applied to prognosis determination. Furthermore, it is possible to provide a screening method for anticancer agents using ESRP expression as an index.

The graph which compared the expression level of (delta) EP1 and SIP1 which are a cancer cell type, EMT induction transcription factor, and the expression level of ESRP in 23 types of breast cancer cells. The graph which compared the expression level of an EMT induction transcription factor other than (delta) EP1 and SIP1, and ESRP in 23 types of breast cancer cells. Figure 5 shows ESRP and δEP1 expression in primary tumor samples from breast cancer patients. This is a graph showing that ESRP expression is suppressed by EMT-induced transcription factor Snail upregulation in a pancreatic cancer cell line that induces luciferase expression by ESRP expression induction. . It shows that EMT induction is suppressed by overexpression of ESRP. It shows that EMT induction is suppressed by overexpression of ESRP.

The form for implementing this invention is demonstrated below.
ESRP expression may be evaluated by mRNA expression level or protein expression level. The detection method of mRNA expression is not particularly limited, and examples thereof include quantitative RT-PCR and in situ hybridization. The method for detecting protein expression is not particularly limited, and examples thereof include immunohistological staining, Western blot method, and ELISA method. The antibody to be used is not particularly limited as long as ESRP is used as an antigen and binds to the antigen, and a mouse antibody, a rat antibody, a rabbit antibody, a sheep antibody, and the like can be appropriately used.

In the type of cancer where the involvement of a specific EMT-inducible transcription factor has been clarified, in addition to using suppression of ESRP expression as a diagnostic indicator, diagnosis is also performed with increased expression of that specific transcription factor This makes it possible to perform a diagnosis with higher accuracy. For example, in breast cancer, a combination of increased EMT-induced transcription factors δEF-1 and / or SIP1 and suppression of ESRP can be used as a diagnostic indicator. In pancreatic cancer, EMT-induced transcription factor Snail increases and ESRP suppression. Can be used as a diagnostic index.

Moreover, if suppression of the expression of ESRP can be suppressed, it will lead to suppression of EMT acquisition. Therefore, it can be applied to a screening method for anticancer agents using the expression level of ESRP as an index. Specifically, anticancer drug candidate substances may be administered to cancer cells or tissues, and then ESRP expression may be detected, resulting in an increase in ESRP expression levels before administration of the candidate substance. If confirmed, the possibility that the candidate substance can be an anticancer drug can be suggested.

Applicable cancers include all solid tumors where EMT acquisition is an indicator of malignancy.
Examples of the present invention will be described below, but the present invention is not limited to the following examples.

[Expression of ESRP in breast cancer cell lines (examination by mRNA expression)]
FIG. 1 shows a graph in which the expression level of ESRP mRNA in 23 breast cancer cell lines with different malignancy levels was evaluated using quantitative RT-PCR. Simultaneously with the expression level of ESRP, the expression levels of EMT-induced transcription factors δEF1 and SIP1 were also examined.

Total RNA was extracted using RNeasy Mini Kit (QIAGEN). RNA was prepared using random primers and ^{Primescript 2 1 st strand cDNA Synthesis kit} (TaKaRa). Quantitative RT-PCR was performed using ABI PRISM 7500 Fast Real-Time PCR System (Applied Biosystems) and Power SYBR Green (Roche Diagnostics).

As a result, Luminal breast cancer cells, which are known to have low malignancy, showed low expression of EMT-induced transcription factors δEF1 and SIP1 and high ESRP expression. On the other hand, Basal breast cancer cells, which are known to have high malignancy, showed high expression of EMT-inducible transcription factors δEF1 and SIP1 and low ESRP expression. Therefore, it was shown that malignancy can be diagnosed by using suppression of ESRP expression as an index.

FIG. 2 shows the results of examining the expression levels of the same breast cancer cell line as in FIG. 1 for the three EMT-inducing transcription factors Twist, Snail, and Slung other than δEF1 and SIP1. Unlike δEF1 and SIP1, Twist, Snail, and Slung did not correlate with the malignancy of breast cancer. From the above, in the diagnosis of malignancy in breast cancer, a more accurate diagnosis can be performed by combining the suppression of the expression level of ESRP and the increase in the expression levels of ΔEF1 and SIP1.

[ESRP expression in breast cancer tissues (examination based on protein expression level)]
FIG. 3 shows the result of immunohistochemical staining of a tissue sample of a primary tumor collected from a breast cancer patient. HE shows hematoxylin and eosin staining, and is performed to understand cell morphology. ESRP and cytokeratin 19 (K19; a component that forms the cytoskeleton. When the cells change to EMT-acquired cells, the cytoskeleton is lost.) Were detected in the tumor follicles. On the other hand, δEF-1 of the EMT-inducible transcription gene was shown to be expressed in stromal cells and spindle cells transformed by tumor follicle cells. Therefore, it was shown that the expression level of ESRP can be used as a negative index for EMT acquisition, and that δEF-1 can be used as a positive index for EMT acquisition in breast cancer.

The tissue used for immunohistological staining was fixed with formalin and paraffin sections. In addition, anti-ESRP1 antibody is a mouse monoclonal antibody from Sigma-Aldrich.
The 19 antibody was a rabbit monoclonal antibody purchased from Epitomics, and the anti-δEF1 antibody was a rabbit monoclonal antibody purchased from Novus Biologicals.

[Studies in pancreatic cancer]
We investigated whether Snail, an EMT-inducible transcription factor, suppresses ESRP expression in pancreatic cancer. As shown in FIG. The pancreatic cancer cell line is Panc-1 cells, and the ESRP2-Luc gene encoding luciferase is introduced into the cells downstream of ESRP. Using this, ESRP expression induction was examined by luciferase expression induction. As a result, it was shown that Snail is effective as an EMT-induced transcription factor in pancreatic cancer, and that ESRP expression decreases simultaneously with Snail expression. Luciferase activity was evaluated using luciferase reporter assay system (Promega) and AutoLumat LB953 (EG & G Berthold) as a luminometer.

[Inhibition of EMT acquisition by ESRP]
ESRP1 and ESRP2 were forcibly expressed in MDA-MB-231 cells (see FIG. 1) with high malignancy and cultured. As shown in FIG. 5, in the cells in which ESRP1 and ESRP2 were forcibly expressed as compared with the control cells, the increase in the value of E-cadherin, which is a characteristic of epithelial cells, was measured at the mRNA level (assessed by quantitative RT-PCR). And at the protein level (evaluated by immunoblotting).

Moreover, it is known that transformation to EMT is induced by TGF-β administration, but ESRP1
It was examined whether or not the transformation of EMT by TGF-β occurred by forcibly expressing ESRP2 and ESRP2. The result is shown in FIG. As shown in the immunofluorescence detection method, by forced expression of ESRP1 and ESRP2, E-cadherin, which is a feature of epithelial cells, is detected in cells administered with TGF-β, and transformation to EMT is suppressed. It was shown that.

  From the above, it was shown that the promotion of malignancy can be suppressed by increasing the expression level of ESRP. That is, it is possible to screen for anticancer drugs using the increase in the expression level of ESRP as an index.

The immunoblotting and immunofluorescence detection methods were performed by commonly used methods, and details of the immunofluorescence detection methods are as follows. PBS with 3.7% formaldehyde in PBS
For 15 minutes, placed in PBS containing 0.2% Triton X-100 for 5 minutes, and then the primary antibody dissolved in Blocking One Solution (Nacali) was allowed to act overnight at 4 ° C. Then add the secondary antibody to 1
It was allowed to act for a period of time, and TOTO3 (Invitrogen-Molecular Probe) was allowed to act for 5 minutes. Then, it detected using the confocal laser microscope. The anti-E-cadherin antibody used for immunoblotting was purchased from BD Transduction Laboratories.

Claims (6)

  A method for detecting the malignancy of breast cancer cells using as an index a combination of the suppression of ESRP expression and the increase of EMT-inducible transcription factor δEF-1 and / or SIP1 in cancer samples .   A method for detecting the malignancy of pancreatic cancer cells, which uses as an index a combination of suppression of ESRP expression and an increase in EMT-induced transcription factor Snail in cancer samples.   A method for screening an anticancer drug, wherein a candidate substance is administered to a cancer cell or cancer tissue in vitro, and then the expression of ESRP and the expression of EMT-induced transcription factor δEF-1 or SIP1 are detected, A method for screening an anticancer drug for breast cancer, wherein the expression level of ESRP is increased and the expression level of δEF-1 or SIP1 is decreased compared to before administration of the candidate substance.   A screening method for anticancer drugs, in which a candidate substance is administered to cancer cells or tissues in vitro, and then ESRP expression and EMT-induced transcription factor Snail are detected before the candidate substance is administered. A method for screening an anticancer drug for pancreatic cancer, using as an index the increase in the expression level of ESRP and the decrease in the expression level of Snail.   A method for detecting the malignancy of breast cancer cells, characterized in that the lower the expression of ESRP and the higher the expression of EMT-inducible transcription factor δEF-1 or SIP1, the higher the malignancy of breast cancer in a cancer sample. A method for detecting the malignancy of pancreatic cancer cells, characterized in that the lower the expression of ESRP and the higher the expression of EMT-inducible transcription factor Snail, the higher the malignancy of pancreatic cancer in a cancer sample.