Pancreatic cancer markers

Abstract

Traditionally, CA19-9 was used as a marker for detecting pancreatic cancer, but some patients did not secrete CA19-9 at all. Therefore, there was a need for a substance that is directly secreted by the pancreas and can serve as a marker for pancreatic cancer, as an alternative to CA19-9. [Solution] It was confirmed in vovo that pancreatic cells suppress the secretion of fibulin-1 when they become cancerous. In other words, a marker for detecting pancreatic cancer, characterized by being composed of fibulin-1, can raise suspicion of pancreatic cancer if a decrease in blood concentration from normal is observed.

Description

Technical Field

[0001] The present invention relates to a marker for detecting pancreatic cancer.

Background Art

[0002] In 2022, the number of newly diagnosed pancreatic cancer patients reached 511,000. In addition, 467,000 pancreatic cancer-related deaths occurred. Pancreatic cancer ranks sixth among cancer-related causes of death worldwide. Moreover, despite improvements in treatment methods, the five-year survival rate of pancreatic cancer patients remains low at 15% or less. Furthermore, even for patients who have successfully undergone treatment, the five-year survival rate is reported to be approximately 20%.

[0003] To improve the survival rate, it is important to detect pancreatic cancer at an early stage. However, since pancreatic cancer has few early symptoms, early detection is difficult. Therefore, the establishment of a method for easily detecting pancreatic cancer has become an important issue.

[0004] Conventionally, CA19-9 has been used as the most specific and sensitive biomarker for detecting pancreatic cancer. However, it has been reported that patients with the Lewis antigen-negative type who do not secrete (or secrete a small amount of) CA19-9 exist at a rate of about 5-10%. Considering this proportion among pancreatic cancer patients, it corresponds to approximately 34%.

[0005] In recent years, inflammatory factors such as lymphocytes and monocytes and C-reactive protein have been attracting attention as useful pancreatic cancer diagnostic biomarker candidates instead of CA19-9 (Non-Patent Documents 1-5).

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

[0007] However, because these candidate pancreatic cancer diagnostic markers are not molecules directly secreted by the pancreas, it is difficult to identify a causal relationship. Furthermore, the actual usefulness of these candidate pancreatic cancer diagnostic markers in diagnosing pancreatic cancer has not yet been clarified. Therefore, identifying novel candidate molecules that can serve as pancreatic cancer diagnostic markers among molecules directly secreted by pancreatic cancer cells could lead to the development of early detection methods and improved prognosis. [Means for solving the problem]

[0008] This invention aims to solve the above problems. By comprehensively analyzing proteins expressed in normal pancreatic duct cells and pancreatic cancer cells using shotgun proteomics with liquid chromatography (LC)-mass spectrometry (MS), and identifying proteins whose expression is specifically altered in pancreatic cancer cells, the invention was completed.

[0009] More specifically, the marker for detecting pancreatic cancer according to the present invention is characterized by comprising fibulin-1. [Effects of the Invention]

[0010] Fibulin-1 (which may also be called "fibrin-1") is a secretory protein expressed in both normal pancreatic duct cells and pancreatic cancer cells. Furthermore, its expression is altered in pancreatic cancer cells compared to normal pancreatic cells, making it a protein directly secreted by the pancreas. Moreover, when pancreatic cells become cancerous, its expression changes significantly, and this change is reflected in its blood concentration. Therefore, pancreatic cancer can be detected at the level of a blood test, making it easy to detect.

[0011] Furthermore, since fibulin-1 is independent of CA19-9 secretion, pancreatic cancer can be detected even in Lewis antigen-negative types. [Brief explanation of the drawing]

[0012] [Figure 1] This Venn diagram shows the results of shotgun proteomics, performed to comprehensively analyze proteins expressed in normal pancreatic ductal cells and pancreatic cancer cells, broken down by cell type. [Figure 2] This is a list of proteins identified by semi-quantitative analysis based on spectral counting as proteins whose expression is commonly altered in pancreatic cancer cells, BxPC-3 and PANC-1. [Figure 3] This is a list of proteins identified by semi-quantitative analysis based on spectral counting as proteins whose expression is commonly altered in pancreatic cancer cells, BxPC-3 and PANC-1. [Figure 4] This is a list of proteins identified by semi-quantitative analysis based on spectral counting as proteins whose expression is commonly altered in pancreatic cancer cells, BxPC-3 and PANC-1. [Figure 5]A list of proteins identified as proteins whose expression levels vary commonly in pancreatic cancer cells BxPC-3 and PANC-1 by semi-quantitative analysis based on spectral counts. [Figure 6] A list of proteins identified as proteins whose expression levels vary commonly in pancreatic cancer cells BxPC-3 and PANC-1 by semi-quantitative analysis based on spectral counts. [Figure 7] A graph of proteins identified as "intracellular components" by DAVID through gene ontology (GO) analysis of pancreatic cancer-related proteins and their expression levels. [Figure 8] A graph of proteins identified as "molecular functions" by DAVID through gene ontology (GO) analysis of pancreatic cancer-related proteins and their expression levels. [Figure 9] A graph of proteins identified as "biological processes" by DAVID through gene ontology (GO) analysis of pancreatic cancer-related proteins and their expression levels. [Figure 10] A graph of proteins identified as "pathways" by DAVID through gene ontology (GO) analysis of pancreatic cancer-related proteins and their expression levels. [Figure 11] A list of 25 proteins classified into the "extracellular region" that may be detected in the blood in the classification of "intracellular components". [Figure 12] A photograph showing the results of Western blot performed to verify whether the expression of fibulin-1 is decreased in pancreatic cancer cells. [Figure 13] A graph representing the results of quantifying fibulin-1 secreted into the culture medium to clarify whether the secretion of fibulin-1 decreases due to pancreatic cancer. [Figure 14] A list of patients who measured the concentration of fibulin-1 in the sera of pancreatic cancer patients. [Figure 15] The measurement results of the concentration of fibulin-1 in the sera of pancreatic cancer patients. [Figure 16]This graph shows the results of a study comparing fibulin-1 expression in pancreatic tissue between healthy individuals and pancreatic cancer patients. [Figure 17] This study investigated the relationship between fibulin-1 expression levels and prognosis. [Figure 18] This study compares the expression levels of fibulin-1 in various cancer patients. [Modes for carrying out the invention]

[0013] The following describes the marker for detecting pancreatic cancer according to the present invention, with reference to examples and drawings. The following description illustrates one embodiment and one example, and the present invention is not limited to the following description. The following description may be modified without departing from the spirit of the invention. Furthermore, embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included within the technical scope of the present invention. All references cited herein are incorporated herein by reference. In this specification, when a numerical range is described as "A to B," it means "A or greater and B or less."

[0014] In this invention, as shown in the following examples, shotgun proteomics using LC-MS was performed to identify candidate biomarkers for pancreatic cancer diagnosis. As a result, 142 candidate proteins with expression levels fluctuating by more than twofold were successfully identified. Furthermore, by utilizing bioinformatics methods based on GO analysis, fibulin-1, a candidate protein detectable in blood, was identified.

[0015] Verification using cultured pancreatic cancer cells suggested that extracellular secretion of fibulin-1 decreases with malignant transformation. Supporting this result, fibulin-1 concentrations in the serum of pancreatic cancer patients were lower compared to healthy individuals. Therefore, a decrease in blood fibulin-1 concentration is considered a useful diagnostic marker for pancreatic cancer.

[0016] Fibulin-1 is a member of the fibulin family, localized on chromosome 22q13.31, and is a secreted glycoprotein that interacts with extracellular matrix (ECM) proteins. Fibulin-1 is known to regulate cell adhesion, extension, and migration by binding to ECM proteins such as fibronectin and proteoglycans. Furthermore, fibulin-1 has been suggested to be involved in regulating intracellular signaling cascades by controlling the actin-myosin motor complex through the inhibition of ERK activity and heavy chain myosin phosphorylation.

[0017] The antitumor effects of fibulin-1 have been reported in various cancers, including gastric cancer, prostate cancer, and estrogen-dependent cancers. On the other hand, there are also reports on its oncogenic function. Recently, Aksoy et al. reported on the function of fibulin-1 in pancreatic cancer based on immunohistochemistry. Similar to the results of this study based on the TCGA database, there was no difference in fibulin-1 expression between pancreatic cancer patients and controls.

[0018] On the other hand, fibulin-1 expression in the microenvironment surrounding pancreatic cancer was associated with cancer progression, with a significantly lower survival rate in the group with high fibulin-1 expression compared to the group with low expression. As shown in the following examples, fibulin-1 levels in the serum of pancreatic cancer patients were significantly lower than in healthy individuals, suggesting that the accumulation of fibulin-1 in the pancreatic cancer microenvironment may have led to a decrease in serum fibulin-1. However, further investigation is needed into the mechanism by which fibulin-1 levels decrease in the serum of pancreatic cancer patients.

[0019] Several studies have investigated the usefulness of fibulin-1 as a biomarker by examining its expression in the blood. The concentration of fibulin-1 in the plasma of patients with papillary thyroid carcinoma was significantly lower than in healthy individuals. Furthermore, the concentration of fibulin-1 in the serum of colorectal cancer patients was significantly lower than in healthy individuals and patients with benign polyps.

[0020] Similar to the results of this analysis based on the TCGA database, it was suggested that decreased fibulin-1 expression in other cancers leads to decreased fibulin-1 levels in the blood. It is known that fibulin-1 mRNA has four different isoforms (fibulin-1A-D) in its C-terminal region due to alternative splicing. Therefore, focusing on the isoforms of fibulin-1 in the blood may improve the specificity of pancreatic cancer diagnosis.

[0021] Furthermore, as shown in the following examples, we were able to identify 25 potential pancreatic cancer biomarkers that can be detected in the blood. We also confirmed that fibulin-1 concentrations were lower in the serum of pancreatic cancer patients compared to healthy individuals. Therefore, changes in blood fibulin-1 concentration are suggested to be useful as a biomarker for diagnosing pancreatic cancer.

[0022] The marker for detecting pancreatic cancer according to the present invention (which may also be called a pancreatic cancer screening marker or simply a pancreatic cancer marker) measures the concentration of fibulin-1 in a biological sample and compares it to the concentration in healthy individuals (which may be called the "reference value"). If the concentration is lower, pancreatic cancer can be suspected. This can be used as useful evidence to proceed to more detailed examinations. Here, the biological sample includes blood.

[0023] Furthermore, the blood fibulin-1 concentration in healthy individuals was 12.30 ± 1.21 ng / mL in the following experiment. Therefore, pancreatic cancer can be suspected if the fibulin-1 concentration in the biological sample is at least lower than 10 ng / mL. In other words, the above reference value can be set at 10 ng / mL.

[0024] Therefore, the present invention is a pancreatic cancer marker comprising fibulin-1. Furthermore, as described above, the use of the pancreatic cancer marker includes the steps of measuring the concentration (actual amount) of fibulin-1 in a biological sample and comparing the actual amount with a reference value of fibulin-1. This can also be described as the use of fibulin-1 as a pancreatic cancer biomarker. [Examples]

[0025] <Material> Dithiothreitol (DTT), Tris(trishydroxymethylaminomethane), Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and iodoacetamide (IAA) were purchased from Fujifilm Wako Co., Ltd. Other reagents were purchased from Merck Ltd.

[0026] <Cultured cells> Cultured pancreatic cancer cells (BxPC-3 and PANC-1) and immortalized human normal pancreatic duct cells (HPNE) were purchased from American Type Culture Collection (ATCC). BxPC-3 and PANC-1 were cultured in RPMI-1640 medium (Fujifilm Wako Co., Ltd.) supplemented with 10% fetal bovine serum (FBS) at 37°C under 5% CO2 conditions. HPNE was cultured in the culture medium recommended by ATCC.

[0027] The culture medium recommended by ATCC is a complete culture medium consisting of a basal medium comprising 75% glucose-free DMEM (Merck), 2 mM glutamine (Fujifilm Wako), 1.5 g / L sodium bicarbonate (Fujifilm Wako), and 25% Medium M3 base (Incell), to which 5% FBS, 10 ng / mL human recombinant EGF (Thermo), and 1 g / LD-glucose (Fujifilm Wako) are added.

[0028] <Protein sample preparation for proteomics and Western blotting> 5 x 10 5Individual BxPC-3, PANC-1, and HPNE were each seeded in 100 mm dishes and cultured for 72 hours. Subsequently, the cells were lysed with lysis buffer (7 M urea, 2 M thiourea, 5% CHAPS, and 1% Triton X-100). The protein concentration was measured by the Bradford method.

[0029] <Trypsin digestion> Trypsin digestion was performed based on past reports (Non-Patent Documents 6-9). 10 μg of protein obtained from each cell was reduced with 45 mM DTT and 20 mM TCEP, and then alkylated with 100 mM IAA. Subsequently, enzymatic digestion was carried out at 37°C for 24 hours using Trypsin Gold, Mass Spectrometry Grade (Promega Corp). The enzymatic digest was desalted using PepClean C-18 Spin Columns (Thermo) according to the manual.

[0030] <LC-MS / MS analysis> Approximately 2 μg of the desalted peptide sample (the above enzymatic digest) was injected into a peptide L-trap column (Chemicals Evaluation and Research Institute) using an HTS PAL autosampler (CTC Analytics). Subsequently, the injected peptides were separated using a reverse-phase C18 column (L-column, particle size: 3 μm, pore size: 120 Å, inner diameter: 0.2 mm, length: 150 mm; Chemicals Evaluation and Research Institute) with Paradigm MS4 (AMR).

[0031] For the mobile phase, Solution A (0.1% formic acid solution) and Solution B (acetonitrile) were used, and gradient elution was performed with the composition of Solution B changed from 5% to 40% over 120 minutes, and the flow rate was set to 1 μL / min for elution. The eluted peptides were analyzed using an LTQ ion trap mass spectrometer (Thermo).

[0032] The analysis results were searched against the SwissProt Homo Sapiens database using Mascot version 2.5.01 (Matrix Science). The search criteria were set as follows: digestive enzyme; trypsin (acceptable value: number of undigested peptide sites ≤ 2), precursor ion mass error; ±2.0 Da, product ion mass error; ±0.8 Da, chemical modification; cysteine ​​carbamide methylation and methionine oxidation.

[0033] <Spectral counting method> To compare the expression levels of the identified proteins, semi-quantitative analysis was performed using the spectral count method. The protein expression ratio was calculated as the Rsc value, which is the base-2 logarithm based on the spectral count value. Proteins with an Rsc value of >1 or <-1, corresponding to a twofold or greater change in expression level, were selected as candidate markers.

[0034] <Bioinformatics> To investigate the function of proteins whose expression was altered in pancreatic cancer cells, we performed functional prediction and classification using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) (https: / / david.ncifcrf.gov / tools.jsp). We also analyzed the association between fibulin-1 expression in cancer tissue and the prognosis of pancreatic cancer patients using the UALCAN database (http: / / ualcan.path.uab.edu).

[0035] <Western blot> Total protein (10 μg) was mixed with loading buffer and heated at 95°C for 10 minutes. After separation by SDS-PAGE (10% gel), the mixture was transferred to a PVDF membrane (Merck) at 15V for 30 minutes. The membrane was blocked with TBS + 0.1% Tween-20 buffer (TBS-T) containing 5% skim milk at room temperature for 2 hours, and then incubated overnight at 4°C with anti-fibulin-1 antibody (1:1,000; abcam). Subsequently, the membrane was washed with TBS-T and incubated with HRP-labeled anti-rabbit IgG antibody (1:4,000; American Qualex) at room temperature for 1 hour.

[0036] Finally, the membrane was reacted with SuperSignal West Dura Extended Duration substrate (Thermo), and the resulting luminescence was imaged using myECL (Thermo). Next, the membrane was stripped with Restore Western Blot Stripping buffer (Thermo), and then incubated overnight at 4°C with anti-β-actin antibody, which served as a loading control.

[0037] <elisa> Fibulin-1 in the culture medium and serum was quantified using the Enzyme-linked Immunosorbent Assay Kit For Fibulin 1 (Cloud-Clone) according to the manual.

[0038] <Pancreatic cancer patients and serum> Serum samples (n=10) from pancreatic cancer patients were collected at Kansai Medical University Hospital from May 2022 to June 2023. The serum was collected after approval from the ethics committees of Kansai Medical University Hospital (Approval Number: 2021279) and Kinki University Faculty of Pharmaceutical Sciences (Approval Number: 22-197), and after obtaining informed consent from all patients. Serum samples (n=6) from healthy individuals were purchased from KAC Corporation.

[0039] <Statistical analysis> Data are presented as mean ± standard error, and at least three replication experiments were performed. Statistical analysis was performed using GraphPad Prism version 8.1.2 (GraphPad Software). Student's Test was used to determine significant differences between groups, and a P < 0.05 result was considered statistically significant.

[0040] ≪Results≫ <Identification of proteins whose expression is altered in pancreatic cancer cells> Shotgun proteomics was performed to comprehensively analyze the proteins expressed in normal pancreatic duct cells and pancreatic cancer cells. Under the conditions examined, 724 proteins were identified from immortalized normal pancreatic duct cells (HPNE), and 761 proteins were identified from pancreatic cancer cells BxPC-3 and 776 proteins from PANC-1, respectively. The distribution of proteins identified from normal pancreatic duct cells and pancreatic cancer cells is shown in Figure 1 as a Venn diagram.

[0041] Next, semi-quantitative analysis based on spectral counting was performed to identify proteins whose expression was altered in pancreatic cancer cells. Here, positive and negative Rsc values ​​indicate increased and decreased expression in pancreatic cancer cells, respectively. 484 proteins with altered expression were identified from BxPC-3, and 273 proteins from PANC-1. Of these altered-expression proteins, 142 proteins that showed altered expression in both cell types were identified as pancreatic cancer-related proteins. The identified proteins are shown in Figures 2 to 6.

[0042] <Functional Prediction Classification of Pancreatic Cancer-Related Proteins> To explore candidate marker proteins for pancreatic cancer diagnosis, we performed gene ontology (GO) analysis of pancreatic cancer-related proteins. In this study, we used DAVID to analyze "intracellular components" (Figure 7), "molecular function" (Figure 8), "in vivo action" (Figure 9), and "pathways" (Figure 10). In each figure, the horizontal axis represents the assigned protein species, and the vertical axis represents the relative abundance (%). Only proteins in significant categories (p<0.05) are shown.

[0043] As a result, we decided to focus on 25 proteins classified as "extracellular regions" that could potentially be detected in the blood, based on the classification of intracellular components (Figure 11). From these 25 candidates, we focused on the decreased expression of fibulin-1 (indicated by an arrow in Figure 11), whose function in pancreatic cancer has not been clarified.

[0044] <Fibulin-1 expression in cultured cells> Western blotting was performed to verify whether fibulin-1 expression was reduced in pancreatic cancer cells. When fibulin-1 expression was examined in cultured cells, it was found to be significantly reduced in pancreatic cancer cells (BxPC-3 and PANC-1) compared to normal pancreatic ductal cells (HPNE) (Figure 12).

[0045] Next, to determine whether fibulin-1 secretion decreases as a result of developing pancreatic cancer, fibulin-1 secreted into the culture medium was quantified. The results are shown in Figure 13. In Figure 13, the horizontal axis represents cell type and the vertical axis represents fibulin-1 expression concentration (ng / ml). Referring to Figure 13, it was confirmed that the fibulin-1 concentration tended to be lower in pancreatic cancer cells (BxPC-3 and PANC-1) than in normal pancreatic cells (HPNE).

[0046] <Fibulin-1 expression in pancreatic cancer patients> To investigate whether fibulin-1 is suitable as a biomarker for diagnosing pancreatic cancer, the concentration of fibulin-1 in the serum of pancreatic cancer patients was measured. Detailed information about the patients used in the study, such as age and TMN classification, is shown in Figure 14. Referring to Figure 14, in the [Gender] column, "F" represents female and "M" represents male. [pTNM] is the TNM classification, which represents the stage of tumor progression and is a pathological TNM classification. The [CEA (Carcinoembryonic Antigen)] column shows the concentration of the tumor marker (ng / ml).

[0047] The results of fibulin-1 concentration measurements are shown in Figure 15. Referring to Figure 15, the horizontal axis represents patient type ("Normal" represents healthy individuals), and the vertical axis represents fibulin-1 concentration (ng / ml). The results of this measurement showed that the fibulin-1 concentration in the serum of pancreatic cancer patients (9.02 ± 0.93 ng / mL, Pancreatic cancer) was significantly lower than the serum concentration in healthy individuals (12.30 ± 1.21 ng / mL, Normal).

[0048] Next, we used the UALCAN (The University of ALabama at Birmingham CANcer data analysis Portal) database, based on the TCGA (The Cancer Genome Atlas) database, to investigate the association between fibulin-1 expression and prognosis in pancreatic cancer patients.

[0049] Figure 16 shows the expression of fibulin-1 in pancreatic tissue. The horizontal axis represents the distinction between healthy individuals and pancreatic cancer patients in samples from TCGA. The vertical axis represents TPM (Transcript per million). According to the results in Figure 16, there was no difference between healthy individuals (Normal) and pancreatic cancer patients (Primary tumor).

[0050] Figure 17 shows the relationship between fibulin-1 expression levels and prognosis. In Figure 17, the horizontal axis represents prognosis days, and the vertical axis represents survival probability. High expression is defined as fibulin-1 expression levels of 170.945 or higher, while low expression is defined as levels below 170.945. According to Figure 17, no association was found between fibulin-1 expression levels and prognosis.

[0051] Figure 18 compares fibulin-1 expression levels in various cancer patients. Referring to Figure 18, the horizontal axis represents cancer type, and the vertical axis represents expression level. Each cancer group is separated by the vertical axis. In each cancer group, the right side (indicated by a triangle) represents cancer patients, and the left side represents healthy individuals. In cancer patients, cases where fibulin-1 expression is higher in cancer patients than in healthy individuals are indicated by a white triangle. Comparing fibulin-1 expression in healthy individuals (Normal) and cancer patients (Tumor), a tendency for lower fibulin-1 expression was observed in cancer patients compared to healthy individuals across various cancers. [Industrial applicability]

[0052] The biomarker according to the present invention is useful in the early detection of pancreatic cancer, which is considered difficult to detect early.< / elisa>

Claims

1. A marker for detecting pancreatic cancer, characterized by being composed of fibulin-1.

2. Use of fibulin-1 as a pancreatic cancer biomarker.

3. The use of a pancreatic cancer marker includes the steps of measuring the actual amount of fibulin-1 in a biological sample and comparing the actual amount with a reference value of fibulin-1.

Images