Biomarker composition for diagnosing and treating resistance to pancreatic cancer therapeutic agent, and use thereof

Abstract

The present invention identifies the expression mechanism of an RAB10 or MYH9 protein in a cell plasma membrane of a patient having resistance to a pancreatic cancer therapeutic agent. The present invention provides an RAB10 or MYH9 protein as a biomarker for diagnosing the resistance of a pancreatic cancer patient to a therapeutic agent, and provides a pharmaceutical composition for mitigating resistance to a therapeutic agent for pancreatic cancer through the use of an anti-oligonucleotide that inhibits the RAB10 or MYH9 protein.

Description

Biomarker composition for diagnosing and treating resistance to pancreatic cancer treatment and use thereof

[0001] The present invention relates to a biomarker composition for diagnosing and treating resistance to a pancreatic cancer treatment agent (anticancer drug), and more specifically, to identify the mechanism of occurrence of resistance to a pancreatic cancer treatment agent (anticancer drug), and to discover a protein expressed in a specific manner in the cell plasma membrane of a patient with resistance, and to use this for diagnosing and treating resistance to a pancreatic cancer treatment agent.

[0002] This invention was conducted as part of the research project "Discovery of anticancer drug-resistant pancreatic cancer biomarkers using single-cell transcriptome analysis and artificial intelligence techniques" (Project ID: 1711191548, Performing organization: Yonsei University, Research period: 2023.03.01 ~ 2024.02.29) of the Individual Basic Research (MSIT) supported by the Ministry of Science and ICT.

[0003] The pancreas is an organ with an exocrine function that secretes digestive enzymes that break down carbohydrates, fats, and proteins in the food we eat, and an endocrine function that secretes hormones such as insulin and glucagon that regulate blood sugar levels.

[0004] Pancreatic cancer is a mass (tumor) formed by cancer cells in the pancreas. It generally refers to pancreatic ductal adenocarcinoma, but other types include cystic carcinoma and endocrine tumors. Because pancreatic cancer has no specific early symptoms, it is difficult to detect early. The pancreas is thin, only about 2 cm thick, and is surrounded by a capsule. Furthermore, it is closely connected to the superior mesenteric artery, which supplies oxygen to the small intestine, and the portal vein, which carries nutrients absorbed from the intestine to the liver, making it prone to cancer invasion. It also has the characteristic of early metastasis to the nerve bundles and lymph nodes on the back of the pancreas. Pancreatic cancer cells are particularly rapid in growth. The average survival time is 4 to 8 months, and the prognosis is poor, with a 5-year survival rate of 17 to 24% in those who are eligible for surgery.

[0005] Among the many drug therapies used to treat pancreatic cancer, gemcitabine has been the most commonly used treatment for pancreatic cancer patients for the past 20 years. Combination therapy with gemcitabine and other drugs has been used in pancreatic cancer, but the effects have been minimal and have not significantly improved the survival rate of pancreatic cancer patients. Later, FOLFIRINOX (hereinafter referred to as FOLFIRINOX) was developed. It consists of a combination of four drugs: 5-FU (5-fluorouracil), leucovorin, irinotecan, and oxaliplatin. It increased the median survival rate in pancreatic cancer compared to gemcitabine alone. Similarly, the combination of albumin-bound paclitaxel and gemcitabine also showed an effect of increasing the median survival rate in pancreatic cancer compared to gemcitabine alone. However, the use of these treatments (anticancer drugs) can induce resistance in pancreatic cancer patients, so diagnosis of resistance and appropriate combination therapy are necessary.

[0006] The present invention has been conceived to solve the above problems and meet the above needs, and the purpose of the present invention is to provide a biomarker for diagnosing resistance to a therapeutic agent (anticancer agent) used in the treatment of pancreatic cancer.

[0007] Another object of the present invention is to provide a pharmaceutical composition for preventing, suppressing, or alleviating resistance to a therapeutic agent (anticancer agent) used in the treatment of pancreatic cancer.

[0008] According to the present invention, a biomarker composition for diagnosing resistance to a therapeutic agent (anticancer agent) in a pancreatic cancer patient is provided, comprising the RAB10 or MYH9 protein. In the present invention, the RAB10 or MYH9 protein can be detected from a sample of a pancreatic cancer patient, and most preferably, can be detected in the plasma membrane of cells of the pancreatic cancer patient.

[0009] In the present invention, the therapeutic agent (anticancer agent) is not limited to a drug used to treat pancreatic cancer, but most preferably refers to FOLFIRINOX.

[0010] The present invention also provides a kit for diagnosing therapeutic agent resistance in a pancreatic cancer patient, comprising an antibody or peptide that specifically binds to a RAB10 protein, a MYH9 protein, or an immunogenic fragment thereof.

[0011] In another embodiment of the present invention, a composition for alleviating or treating resistance to a therapeutic agent (anticancer agent) in a pancreatic cancer patient is provided, comprising a RAB10 protein expression inhibitor. In the present invention, the RAB10 protein expression inhibitor may be used without limitation as long as it can attenuate the expression level of the RAB10 protein. However, most preferably, the RAB10 protein expression inhibitor refers to an antisense oligonucleotide that specifically binds to the RAB10 protein.

[0012] The present invention also provides a method for providing information for diagnosing resistance to a pancreatic cancer treatment agent (anticancer drug), comprising the steps of: (a) obtaining a biological sample from a subject; (b) measuring the expression level of RAB10 protein in the sample to obtain data on the expression level of RAB10 protein; (c) comparing the expression level of RAB10 protein of the subject obtained in step (b) with the expression level of RAB10 protein of a non-resistant group of the treatment agent (anticancer drug); and (d) determining that resistance to the treatment agent (anticancer drug) exists when the expression level of the RAB10 protein of the subject is higher than the expression level of the RAB10 protein of the non-resistant group.

[0013] In one embodiment, the biological sample may be obtained from an animal, for example, a mammal, or a human, and for example, the biological sample may be isolated from a pancreatic cancer patient. The biological sample may be pretreated prior to the detection step, and may be subjected to pretreatment steps such as homogenization, filtration, distillation, extraction, concentration, inactivation of interfering components, and addition of reagents.

[0014] In the present invention, a "biological sample" or "sample" may mean blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens, tissue cultures, and / or cells derived therefrom, and may be isolated from an individual (e.g., an individual with cancer and / or an individual likely to develop pancreatic cancer). The biological sample may be, for example, tissue, extract, cell lysate, whole blood, plasma, serum, saliva, ocular fluid, cerebrospinal fluid, sweat, feces, urine, milk, ascites fluid, synovial fluid, peritoneal fluid, etc. In one embodiment, the biological sample may be pancreatic tissue, pancreatic cells, and / or a culture of the cells. Preferably, the biological sample may be pancreatic cancer tissue, but is not limited thereto as long as it is a sample derived from a pancreatic cancer patient from which a biomarker for diagnosing resistance to a therapeutic agent (anticancer drug) according to the present invention can be detected.

[0015] In the step (d) of the present invention, if the expression level of the RAB10 protein of the subject is 5 to 10 times higher than the expression level of the RAB10 protein of the non-resistant group, it can be determined that there is resistance to the therapeutic agent (anticancer agent).

[0016] In one embodiment of the present invention, "high level of biomarker" may mean that the concentration of biomarker in the sample or the number of cells expressing the biomarker is about 1.1 times or more, about 1.2 times or more, about 1.3 times or more, about 1.5 times or more, about 1.8 times or more, about 2 times or more, about 2.5 times or more, about 3 times or more, about 3.5 times or more, about 4 times or more, about 4.5 times or more, or about 5 times or more compared to a comparison sample (control sample). Preferably, it means about 5 to 15 times or more, and more preferably, about 5 to 10 times or more.

[0017] In one embodiment of the present invention, “low level of biomarker” may mean that the concentration of the biomarker in the sample or the number of cells expressing the biomarker is about 1.1 times or more, about 1.2 times or more, about 1.3 times or more, about 1.5 times or more, about 1.8 times or more, about 2 times or more, about 2.5 times or more, about 3 times or more, about 3.5 times or more, about 4 times or more, about 4.5 times or more, or about 5 times or less compared to a comparison sample (control sample).

[0018] The above subject (individual, subject) may be an individual suspected of having pancreatic cancer and / or an individual having pancreatic cancer. The subject of the present invention includes any mammal, and the mammal includes not only humans and primates, but also livestock such as cows, pigs, sheep, horses, dogs, and cats.

[0019] As used herein, the terms "diagnose", "diagnosing", "diagnosis" and variations thereof refer to the discovery, determination or recognition of a health condition or situation of an individual based on one or more signs, symptoms, data or other information relating to the individual.

[0020] The terms "diagnose," "diagnosing," and "diagnosis" above, with respect to a specific disease or condition, include the initial detection of the disease; the characterization or classification of the disease; the detection of the progression, remission, or recurrence of the disease; and the detection of the disease response after the administration of treatment or therapy to an individual. Diagnosing resistance to a treatment for pancreatic cancer involves distinguishing individuals who have developed resistance from those who do not have resistance.

[0021] As used herein, the terms "prognose", "prognosing", "prognosis" and variations thereof refer to the prediction of the course of a disease or condition in an individual having the disease or condition (e.g., predicting patient survival), and these terms include the assessment of disease response following the administration of a treatment or therapy to the individual.

[0022] As used herein, the terms "evaluate," "evaluating," "evaluation," and variations thereof encompass both "diagnose" and "prognose," and also encompass judgments or predictions about the course of a disease or condition in an individual who does not have the disease, as well as judgments or predictions about the likelihood of a disease or condition recurring in an individual who has apparently been treated for the disease. The term "evaluate" also encompasses assessing an individual's response to treatment, such as, for example, predicting whether an individual will respond favorably to a treatment or will not respond to a treatment (or, for example, will experience toxicity or other undesirable side effects), selecting a treatment to administer to an individual, or observing or discovering an individual's response to a treatment administered to an individual.

[0023] In the present invention, the mRNA detection may be performed by one or more methods selected from the group consisting of RNA sequencing, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), RNase protection assay (RPA), microarray, and northern blotting, according to a conventional method known in the art, but is not limited thereto.

[0024] In this specification, "measuring protein expression level" refers to a process of confirming the presence and expression level of a protein expressed from a disease marker gene in a biological sample in order to diagnose a disease. Preferably, the amount of protein can be confirmed using an antibody that specifically binds to the protein of the gene. Analysis methods for this include, but are not limited to, Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence activated cell sorter (FACS), protein chip, etc.

[0025] According to the present invention, RAB10 binds to the MYH9 protein and induces resistance to therapeutic agents (anticancer drugs) in the plasma membrane of pancreatic cancer patients. Therefore, by assessing the expression levels of RAB10 or MYH9 as biomarkers in pancreatic cancer patient samples, the presence of resistance to therapeutic agents (anticancer drugs) can be determined.

[0026] In addition, the RAB10 expression inhibitor of the present invention, preferably an antisense oligonucleotide of RAB10, can be administered to a patient with resistance to a pancreatic cancer treatment (anticancer drug) and used as a treatment (treatment method) to prevent or alleviate (suppress) resistance to the treatment (anticancer drug).

[0027] The present invention also provides a use of RAB10 protein and MYH9 protein biomarkers for diagnosing resistance to pancreatic cancer treatment (anticancer drug) in a subject, and provides a method for diagnosing resistance to pancreatic cancer treatment (anticancer drug) using RAB10 protein and MYH9 protein.

[0028] FIG. 1a illustrates a schematic diagram of a process for establishing a FOLFIRINOX-resistant AsPC-1 cell line according to one embodiment of the present invention.

[0029] Figure 1b shows the results of confirming the IC50, IC70, and IC80 values ​​of FOLFIRINOX for each drug against established AsPC-1 cells.

[0030] Figures 1c and 1d show the chemical drug response results to FOLFIRINOX of parental cells (hereinafter, P group) and FOLFIRINOX-resistant AsPC-1 cells (hereinafter, R group). Figure 1c shows relative cell viability. Figure 1d shows the results of quantifying the IC50 fold change (n=3) (paired t-test, mean ± SEM, **p<0.01).

[0031] Figures 1e to 1g show the results of purifying plasma membrane (PM) proteins and performing LC-MS / MS for quantitative proteome analysis according to one embodiment of the present invention. Figure 1e shows the process of isolating plasma membrane protein complexes from cells of groups P and R. Figure 1f shows the results of extracting and purifying a plasma membrane fraction using a plasma membrane extraction kit, and confirming the purified plasma membrane fraction by blotting with anti-Na, K-ATPase (PM marker) and anti-aldolase (Cytosolic protein marker). Figure 1g shows the results of electrophoresis separation by spotting 40 μg of purified plasma membrane proteins on 8% SDS-PAGE.

[0032] Figure 1h is a Venn diagram and table distinguishing proteins uniquely enriched in the plasma membrane of the R group of the present invention. Referring to Figure 1h, in the present invention, 24 of the 49 proteins detected by LC-MS / MS (Mascot score >80) are selected as candidates (see Table 2).

[0033] FIG. 1i shows a gene ontology model that classifies 24 protein candidates selected according to function according to one embodiment of the present invention, and FIG. 1j is a Venn diagram showing the overlap between each functional cluster based on the gene ontology model.

[0034] Figures 2a and 2b illustrate the cell surface expression levels of RAB10, MYH9, and MRP1 in groups P and R, according to one embodiment of the present invention. Figure 2a shows the results of cell surface biotinylation analysis blotting, and Figure 2b shows the quantified results (n=3) after repeated experiments. The bottom of Figure 2b shows the quantified fold change in surface expression of each protein comparing groups R and P. (Mean ± SEM, **p < 0.01)

[0035] Figure 2c shows the results of comparing mRNA expression of ABCC superfamily genes in pancreatic cancer patient tissues from groups P and R, according to one embodiment of the present invention. The RNA-seq data in Figure 2c refer to data from GSE169321 in the GEO database (mean ± SEM, **p<0.01).

[0036] Figure 2d shows the results of quantitative PCR analysis confirming the knockdown efficiency of RAB10 mRNA targeting antisense oligonucleotides (ASOs) according to one embodiment of the present invention. mRNA samples were extracted from AsPC-1 cells after transfection with ASOs targeting the 5'-, 3'-, and In- (300 nM, 48 h) of human RAB10 mRNA (control ASO (scrambled)). (Mean ± SEM 470 (n = 3), **p<0.01)

[0037] Figures 2e and 2f show the results of the WST-8 assay evaluating the effect of silencing the RAB10 gene using ASO after treating cells in groups P and R with FOLFIRINOX for 72 hours according to one embodiment of the present invention. Figure 2e shows the dose-response curve for FOLFIRINOX, and Figure 2f shows the fold change of IC50 comparing RAB10 ASO-based gene depletion in groups P and R (n=3). (Mean ± SEM, **p<0.01)

[0038] FIG. 3a is a schematic diagram of an anti-hRAB1-based affinity capture mass spectrometry analysis (LC-MS / MS analysis) process according to one embodiment of the present invention.

[0039] Figure 3b shows the results of obtaining a RAB10 interacting protein complex by performing co-immunoprecipitation (co-IP) using a RAB10 antibody according to one embodiment of the present invention, and then separating 40 μg of purified proteins extracted from the P group and the R group by electrophoresis using 8% SDS-PAGE.

[0040] Figure 3c illustrates proteins specifically associated with RAB10 in group R, as a Venn diagram and table, according to one embodiment of the present invention. The table on the right side of Figure 3c compares the range and intrinsic size of proteins detected in R. In this experiment, 40 of 151 protein peptides detected by LC-MS / MS with an individual ion score >80 (ion score -10*Log(P), p<0.05) were selected as candidates specifically associated with RAB10 in group R. (See Table 5)

[0041] Figure 3d illustrates an ontology model that classifies a set of genes for 40 proteins selected from the R-group-specific binding partners of RAB10 into genetic functions according to one embodiment of the present invention. Figure 3e shows the overlap between each functional cluster of the gene sets for the 40 proteins classified according to one embodiment of the present invention. According to the results of Figure 3e, MYH9 is identified as a key binding partner of RAB10 in modulating FOLFIRINOX-resistance in pancreatic cancer cells.

[0042] Figures 3f and 3g show the results of a Co-IP experiment performed with an anti-RAB10 (rabbit polyclonal) antibody in groups P and R according to one embodiment of the present invention. In this experiment, protein samples were blotted with mouse monoclonal antibodies against human RAB10 and MYH9, and Figure 3f shows the immunoblotting results. Figure 3g is a graph analyzing the immunoblotting results, and it can be confirmed that the protein-protein interaction between MYH9 and RAB10 increased 6.42-fold. (Mean ± SEM, **p<0.01)

[0043] Figure 4 is a schematic diagram of RAB10 / MYH9-driven retention of MRP1 at the plasma membrane of FOLFIRINOX-resistant pancreatic cancer cells according to one embodiment of the present invention. Referring to Figure 4, under normal conditions (left image), RAB10 promotes MRP1 endocytosis for periodic degradation to maintain cellular homeostasis. However, when cells are FOLFIRINOX-resistant (right image), RAB10 is abnormally retained at the cell surface by binding to MYH9 to anchor MRP1 to the plasma membrane of pancreatic cancer cells. Consequently, MRP1-mediated drug efflux contributes to FOLFIRINOX-resistance in these cells.

[0044] Figures 5a to 5f show the results of Kaplan-Meier survival plot analysis of selected genes according to one embodiment of the present invention. The interaction survival correlation between the selected FOLFIRINOX resistance prognostic biomarker and pancreatic cancer patient survival can be estimated using the Kaplan-Meier plot. The log-rank p value for the plot shows the correlation between the mRNA expression levels of genes including KRT19, KRT8, IARS2, MYH9, RAB10, and PKM and patient survival. In each graph, p<0.05 and FDR<10% are considered statistically prognostic. Figure 5a shows the Kaplan-Meier plot results for KRT19, Figure 5b for KRT8, Figure 5c for IARS2, Figure 5d for MYH9, Figure 5e for RAB10, and Figure 5f for PKM.

[0045] Figures 6a and 6b illustrate mRNA expression information of pancreatic cancer FOLFIRINOX-resistance prognostic biomarkers selected according to one embodiment of the present invention. Figure 6a is a graph comparing the mRNA expression of KRT19, KRT8, IARS2, MYH9, RAB10, and PKM in tissues of pancreatic cancer patients in groups P and R. The RNA-seq data in Figure 6a refers to data GSE169321 in the GEO database (paired t-test, mean ± SEM). Figure 6b is a graph comparing the mRNA expression of genes of interest in pancreatic cancer cells in groups P and R, using RNA-seq analysis and quantitative PCR based on RNA samples extracted from groups P and R.

[0046] Figures 7a and 7b show the results of confirming the mRNA expression of ABCC superfamily genes in pancreatic cancer patient tissues and pancreatic cancer cells. Figure 7a shows the results of comparing the mRNA expression of ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, ABCC6, ABCC10, and ABCC11 in the tissues of pancreatic cancer patients in groups P and R. RNA-seq data refers to data GSE169321 in the GEO database (paired t-test, mean ± SEM). Figure 7b shows the results of comparing the mRNA expression of ABCC superfamily genes in pancreatic cancer cells in groups P and R and in the tissues of pancreatic cancer patients. In this experiment, RNA-seq analysis and quantitative PCR were performed based on RNA samples extracted from AsPC-1 cells in groups P and R.

[0047] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. The advantages and features of the present invention, and the embodiments that achieve them, will become clearer with reference to the embodiments described below. However, the present invention is not limited to the embodiments disclosed below, but can be implemented in various different forms. These embodiments are provided only to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims. Like reference numerals designate like elements throughout the specification.

[0048] Unless otherwise defined, all terms (including technical and scientific terms) used herein may be used in a sense commonly understood by those of ordinary skill in the art to which the present invention pertains. Furthermore, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise. The terminology used herein is for the purpose of describing embodiments and is not intended to limit the present invention. In this specification, singular forms also include plural forms, unless specifically stated otherwise.

[0049]

[0050] [Example 1]

[0051] 1-1. Cell culture

[0052] The AsPC-1 pancreatic ductal adenocarcinoma (PDAC) cell line obtained from the Korean Cell Line Bank was maintained in Roswell Park Memorial Institute (RPMI) 1640 medium 1x containing 2.05 mM L-glutamine, 25 mM HEPES, 15 mg / L L-methionine, 2000 mg / L sodium bicarbonate, and 5 mg / L Phenol Red (Welgene #LM011-05, Korea) supplemented with 10% fetal bovine serum (FBS) (Welgene #S101-01, Korea) and 1% 100x antibiotic-antimycotic (100 units / mL penicillin, 100 units / mL streptomycin, 250 ng / mL amphotericin B) (Gibco #15240062, Carlsbad, CA, US). Cells were grown at 37°C in a 5% CO2 incubator.

[0053]

[0054] 1-2. Reagents and antibodies

[0055] Oxaliplatin (Sigma-Aldrich #O9512), irinotecan hydrochloride (Sigma-Aldrich #1347609), fluorouracil (Sigma-Aldrich #F0250000), calcium folinic acid salt hydrate (Sigma-Aldrich #F7878), and dimethyl sulfoxide (DMSO) (Sigma-Aldrich #D2650) were purchased commercially. Additionally, commercially available antibodies include: anti-RAB10 (Cell Signaling Technology #8127), anti-MYH9 (GeneTex #GTX633295), anti-RAB10 (GeneTex #GTX82800), anti-MRP1 / ABCC1 (D5C1X) (Cell Signaling Technology #72202), anti-Na, K-ATPase (Cell Signaling Technology #3010), and anti-Aldolase A (Cell Signaling Technology #3188).

[0056]

[0057] 1-3. WST-8 cytotoxicity assay and IC50 calculation

[0058] The concentration of FOLFIRINOX therapy used in the in vitro experiment was based on the clinical dose, so the 1x concentration of each component was set as Oxaliplatin 0.46 μM (DMSO solvent), Irinotecan 5.7 μM (DMSO solvent), Folinic acid 6.1 μM (H2O solvent), and 5-Fluorouracil 46.3 μM (DMSO solvent). The combination of FOLFIRINOX was serially diluted at 100x concentration, Oxaliplatin (46 μM), Irinotecan (570 μM), Fluorouracil (4630 μM), and Leucovorin (610 μM), respectively. The four drugs were serially diluted 10-fold in the pancreatic ductal adenocarcinoma AsPC-1 cell line. 5x10 4 / well Cells were initially seeded in 96-well plates, and the culture medium was removed and replaced with FOLFIRINOX-containing medium 24 hours later. After culturing with FOLFIRINOX for 72 hours, 10 μL of CCK-8 was added to each well, and cell viability was assessed using the Cell Counting Kit-8 (CCK-8, i.e., WST-8) (Sigma-Aldrich #96992, Inc., St. Louis, MO, USA). After 3 hours of incubation at 37°C, the plates were read at 450 nm / 650 nm. Cell IC50, IC70, and IC80 values ​​were calculated using GraphPad Prism by linear approximation regression of viability versus drug concentration, and the results are shown in Figure 1B (GraphPad Software, Inc., La Jolla, CA, USA).

[0059]

[0060] 1-4. Generation of anticancer drug-resistant pancreatic cancer cell lines

[0061] Establishment of chemotherapy-resistant cancer cells was performed using a slightly modified previously described technique. Pancreatic cancer AsPC-1 cells were treated with the IC50 concentration of FOLFIRINOX. After 72 hours, the culture medium was changed to drug-free medium, and the cells were passaged twice. The initially selected AsPC-1 cells were sequentially exposed to medium containing FOLFIRINOX at the IC70 and IC80 concentrations for the second and third selections, and the first selection procedure was repeated. FOLFIRINOX resistance of the cancer cells was verified by WST-8 cytotoxicity assay after two passages. FOLFIRINOX-resistant cells were treated with the IC80 concentration of FOLFIRINOX every two weeks to prevent reversion to a chemosensitive equilibrium state.

[0062]

[0063] 1-5. Purification of plasma membrane proteins, liquid chromatography-mass spectrometry (LC-MS / MS), and unbiased proteomic analysis.

[0064] Equal amounts of plasma membrane proteins were extracted from parental (P) and FOLFIRINOX-resistant (R) AsPC-1 cells using a plasma membrane extraction kit (Abcam #ab65400, Cambridge, UK) according to the manufacturer's instructions. Purified 40 μg of protein from each plasma membrane sample was applied to 8% SDS-PAGE for 1-D electrophoresis (1-DE). After separation by 1-DE, proteins were fixed and visualized using Coomassie Blue MS-compatible stain. The visualized proteins were divided into seven fractions ranging from 30 to 400 kDa in mass and excised from the gel. Each gel band was washed, dehydrated, and then trypsin-digested using a filter-assisted sample preparation method. Tryptic peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) using a nanoACQUITY-UPLC system (Waters Corp. USA) connected to an LTQ Orbitrap XL hybrid ion trap mass spectrometer (Thermo Scientific, USA) for analysis of the generated peptides, followed by database searching against the human NCBIprot database. In this example, the false discovery rate (FDR) for peptides and proteins was strictly controlled to less than 1%.

[0065] Mascot Probability-Based Scoring was performed after the database search. Peptide-spectrum match identifications were filtered by setting individual ion scores >33 (where ion scores are -10*Log(p), p<0.05). The resulting list of 339 proteins was processed to select protein peptides uniquely detected in the R group. Fifty-five proteins enriched in the plasma membrane of the R group were then narrowed down to five candidates as pancreatic cancer prognostic biomarkers based on The Human Protein Atlas (HPA) (https: / / www.proteinatlas.org / ). Kaplan-Meier plots for pancreatic cancer were also generated using Kaplan-Meier Plotter (https: / / kmplot.com / analysis). The false discovery rate (FDR) was calculated to correct for hypothesis testing, and results were considered significant only when the FDR was <10%. Gene Set Clustering based on Functional Annotation was analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) Gene Ontology Resources (https: / david.ncifcrf.gov / ).

[0066]

[0067] 1-6. Antisense oligonucleotides (ASOs) and transfection

[0068] AsPC-1 cells were plated in 6-well plates at 150,000 cells per well. 300 nM ASO was transfected using ViaFect transfection reagent according to the manufacturer's instructions (Promega #E4981, Madison, US). Antisense oligonucleotides (ASOs) were custom designed and synthesized (Bio Basic Canada Inc.) based on previous literature. RNA collection and cytotoxicity assays were performed after 48 h of culture. The sequences of each ASO are listed in Table 1 below. In Table 1 below, an asterisk (*) indicates phosphorothioate (PS) modification of the internucleotide linkage by replacing a non-bridging oxygen in the oligo backbone with a sulfur atom to render the ASO resistant to nuclease degradation.

[0069] Anti-Sense Oligonucleotides Sequences Sequence Numbers scramble ASOC*C*T*CTTACCTCAGTTACAATTT*A*T*ASEC 15'-hRAB10 ASOT*C*G*TACGTCTTCTTCGC*C*A*TSEC 23'-hRAB10 ASOT*C*A*GCAGCATTTGCTCT*T*C*CSEC 3internal hRAB10 ASOT*C*A*TCTATGTTTCTAAGCCATT*T*G*CSEC 4

[0070]

[0071] 1-7. Reverse transcription (RT-PCR) and quantitative PCR analysis (qPCR)

[0072] Forty-eight hours after transfection with ASO, total RNA was extracted from AsPC-1 cells using the RNeasy Micro Kit (Qiagen #74004, QIAGEN GmbH, Verogen, Inc.) according to the manufacturer's protocol. To reverse transcribe RNA into cDNA, the purified RNA sample was combined with EcoDry premix (Takara Bio Inc. #639549, Shiga, Japan). The mixture was incubated at 42°C for 1 hour and then at 70°C for 10 minutes.

[0073] qPCR was performed using the Applied Biosystem StepOne System (Applied Biosystems, Forster City, CA, 188 USA). Real-time PCR reactions were measured by detecting the binding of fluorescent SYBR Green dye to double-stranded DNA. For PCR amplification, 100 ng cDNA, 2 μL primer set, 10 μL 2x SYBR premix Ex Taq, and 0.4 μL 50x ROX reference dye (Takara #RR420L) were mixed, and the total reaction volume was adjusted to 20 μL with RNase-free water. Amplification was performed under the following cycling conditions: 95°C for 15 min, followed by 40 cycles of 95°C for 15 s and 60°C for 40 s. Analysis was performed in triplicate for each cDNA. Relative mRNA gene expression was normalized to the housekeeping gene GAPDH, and ΔCt values ​​were calculated as follows: ΔCt = Ct(GAPDH) - Ct(target gene). The average ΔCt was then subtracted from each experimental condition described to yield ΔΔCt values. Fold changes in gene expression were normalized to GAPDH and calculated as 2-ΔΔCt relative to the control sample.Primer sequences used in qPCR analysis are as follows: GAPDH, forward primer 5'-AAT CCC ATC ACC ATC TTC CA-3', reverse primer 5'-TGG ACT CCA CGA CGT ACT CA-3'; hRAB10, forward primer 5'-CGA CCT GCT TTT CAA GCT GC-3', reverse primer 5'- AGG CAT CAT CCG AAA AAC GA-3'; hKRT8, forward primer 5'-GGC CGT GGT TGT GAA GAA GA-3', reverse primer 5'- CTT GGG CAG GAC GTC AGA G-3'; hKRT19, forward primer 5'- TGG AGA TGC AGA TCG AAG GC-3', reverse primer 5'- GCC CCT CAG CGT ACT GAT TT-3'; hPKM, forward primer 5'- TCA CGC TGG ATA ACG CCT AC-3', reverse primer 5'- AGA TCT TGC TGC CCA CTT CC-3'; hIARS2, forward primer 5'- AGC CCA AGA GTG TTT TCC GT-3', reverse primer 5'- TCT TCC AAC CCG GGC TTT TT-3.

[0074]

[0075] 1-8. Immunoprecipitation and Affinity Capture Mass Spectrometry

[0076] For affinity capture mass spectrometry, co-immunoprecipitation with anti-RAB10 antibodies was performed. 1500 μg protein lysate was diluted to a final volume of 1000 μL in lysis buffer (20 mM Tris [pH 7.4], 200 mM NaCl, 1% NP40 [v / v], 1 mM EDTA-Na2 supplemented with a protease inhibitor cocktail) and precleared by incubation with normal IgG for 30 min at 4°C and with control agarose resin (cross-linked 4% bead agarose) for 2 h at 4°C. The mixture was then centrifuged at 3,000 rpm for 10 min, and the protein lysate was collected from the supernatant.

[0077] The pre-cleared protein lysate was then incubated overnight at 4°C with RAB10 antibody. The target protein was precipitated by binding to pre-equilibrated 10% protein A / G resin (Thermo Pierce #20421) in lysis buffer at 4°C for 6 h, collecting the immune complexes, which were then washed five times with lysis buffer before elution with 2x SDS-PAGE sample buffer, electrophoresis, and immunoblotting. The purified protein complexes were subjected to LC-MS / MS for quantitative proteomic analysis.

[0078]

[0079] 1-9. Surface biotinylation and immunoblotting

[0080] Surface biotinylation of AsPC-1 cells was performed according to a published method. AsPC-1 cells (1 × 10 6(Dog cells / well) were qodid for 5 min at 4°C and washed three times with chilled phosphate-buffered saline (PBS). The proteins expressed in the plasma membrane of the cultured cells were biotinylated with 1 mL biotin solution (0.3 mg / mL Sulfo-NHS-SS-biotin in cold PBS [Thermo Pierce #21331, Waltham, MA, USA]) for 30 min at 4°C in the dark. Next, the cells were incubated with quenching buffer containing 0.5% bovine serum albumin (BSA) in PBS at 4°C for 10 min to remove excess biotin, and then washed three times with PBS. Subsequently, surface biotinylated cells were harvested in lysis buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 1% (v / v) NP40, 10% glycerol, and 1 mM EDTA-Na2 supplemented with protease inhibitor cocktail (Sigma-Aldrich, Roche #04693159001). Cell lysates were homogenized by sonication for 20 s (1 s pulse) and then centrifuged at 16,000 × g for 20 min at 4°C. The resulting supernatant containing 400 μg of total protein was incubated with 200 μL 10% streptavidin agarose (Thermo Pierce #20349). Streptavidin-bound biotinylated proteins were then centrifuged and washed five times with lysis buffer. Next, biotinylated proteins were eluted in 20 μL 2x SDS sample buffer supplemented with 0.02 g / mL DL-dithiothreitol (DTT) (Sigma-Aldrich #43815) using a vortex mixer (1,000 rpm) at 37°C for 30 min and separated by 4–12% SDS-polyacrylamide gel electrophoresis (Thermo Fisher #NW04120).The separated proteins were electrotransferred to nitrocellulose membranes (GE Healthcare, Amersham #10600004, Chicago, IL, USA) and blotted with appropriate primary and secondary antibodies (5% skim milk). Protein bands were detected by enhanced chemiluminescence (GE Healthcare, Amersham #RPN2105), and the density of each protein band was quantified using imaging software Fiji (ImageJ ver. 2.9.0, NIH, USA).

[0081]

[0082] 1-10. Bulk RNA sequencing analysis

[0083] RNA was extracted from AsPC-1 cells using the RNeasy Micro Kit (Qiagen #74004) for RNA isolation. RNA quality was measured using the RNA 6000 Nano Chip (Agilent Technologies, Inc., CA, USA). mRNA was isolated from total RNA using the Poly(A) RNA Selection Kit (Lexogen, Inc., Austria), and cDNA was synthesized according to the manufacturer's instructions. The library was verified using the Agilent 2100 bioanalyzer (DNA high sensitivity kit) to assess the average fragment size. High-throughput sequencing was performed using the HiSeq 2500 (Illumina, Inc., USA) and paired-end 100 sequencing, followed by quantification. mRNA-seq was imported using the TopHat software tool to obtain the mapped alignment file. The coverage of Bedtools was used to evaluate differentially expressed genes based on the number of unique multiple alignments. Read count data were also used to assemble transcriptomes, estimate abundance, and detect differential expression of genes or isoforms via cufflinks.

[0084]

[0085] 1-11. Statistical Analysis

[0086] The results of several experiments conducted in this invention are expressed as the mean ± the standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA) using one-way ANOVA followed by Tukey's multiple comparison test, as appropriate. In this invention, P<0.05 is considered statistically significant.

[0087]

[0088] [Example 2]

[0089] Results analysis

[0090] 2-1. Confirmation of detection of RAB10 on the surface of anticancer drug-resistant pancreatic cancer cells.

[0091] To understand the role of surface-expressed proteins in chemosensitivity, we selected FOLFIRINOX-resistant AsPC-1 cell lines by sequentially treating cells with the IC50, IC70, and IC80 of FOLFIRINOX (Fig. 1b) in an on- (72 h) and off- (72 h) mode (Fig. 1a). First, the chemosensitivity of the selected cells was tested using the WST-8 cytotoxicity assay, and the IC50 was increased 3.8-fold compared to the parental cells. Therefore, the selected cells were confirmed to be FOLFIRINOX-resistant cells (Fig. 1c and d). Then, the parental and FOLFIRINOX-resistant AsPC-1 cells were harvested, and the plasma membrane proteins were extracted using a plasma membrane isolation kit (Fig. 1e-g), followed by trypsin digestion and liquid chromatography-mass spectrometry (LC-MS / MS) to compare the membrane protein expression levels (Fig. 1h). A total of 339 proteins were identified in the parental cell group (hereinafter referred to as P group) (228 proteins) and the porfirinox-resistant cell group (hereinafter referred to as R group) (111 proteins), and 49 of these proteins were confirmed to be enriched in the plasma membrane fraction of the R group.

[0092] Twenty-four of the 55 protein peptides detected by LC-MS / MS with individual ion scores higher than 80 were selected as candidates for enrichment in the plasma membrane of FOLFIRINOX-resistant AsPC-1 cells. Based on the Human Protein Atlas (HPA) database, five of these 24 proteins were selected as prognostic biomarkers, and high expression of the selected biomarkers was detrimental to the survival of pancreatic cancer patients. Five candidates, including KTR8, KTR19, PKM, RAB10, and IARS2 (Fig. 1h and Table 2), showed statistical significance (p<0.01, FDR<10%) as indicated by the log-rank p value of the Kaplan-Meier plot estimating the correlation between mRNA expression levels and patient survival (Figs. 5a to 5f). mRNA expression of the five candidate genes was confirmed in patient-derived PDAC tissues regardless of FOLFIRINOX treatment (Fig. 6a), and the remaining tumor tissue resected surgically from a patient treated with 8 cycles of neoadjuvant FOLFIRINOX was considered FOLFIRINOX-resistant. Interestingly, selected biomarkers, such as RAB10, were enriched in pancreatic endocrine cells (Table 3, Human Protein Atlas). Nevertheless, total mRNA expression was not significantly altered by FOLFIRINOX resistance in pancreatic ductal adenocarcinoma cells (Fig. 6b, quantitative PCR and RNA-seq).

[0093]

[0094] Protein information for 24 of 339 proteins detected in the plasma membrane fraction of FOLFIRINOX-resistant AsPC-1 cells번호밴드 라벨MW in Gel (kDa)유전자 명칭질량 (kDa)Mascot 점수PDAC 예후1FOL-R_545~50ATP5F1B56.53349NP2FOL-R_545~50KRT1930.81719unfavorable3FOL-R_545~50KRT830.8741unfavorable4FOL-R_360~90IARS287.7553NP5FOL-R_360~90GPD281.3422NP6FOL-R_290~150ACTN1103.6385NP7FOL-R_1200~400DPP488.9371NP8FOL-R_1200~400SLC25A533.1329NP9FOL-R_1200~400MYH9227.7315NP10FOL-R_1200~400SLC25A433.1267NP11FOL-R_360~90HSPA571.0229NP12FOL-R_545~50DLST49.1227NP13FOL-R_360~90ALDH18A188.0195NP14FOL-R_730~40MRPL435.2191NP15FOL-R_730~40TOMM4038.3186NP16FOL-R_545~50DLD54.7147NP17FOL-R_1200~400RAB1022.7131unfavorable18FOL-R_450~60PCK271.5131NP19FOL-R_730~40PON239.5105NP20FOL-R_1200~400ATP2A2111.1101NP21FOL-R_730~40CBX547.1100NP22FOL-R_1200~400KRT3148.696NP23FOL-R_3200~400IGKV3-1111.894NP24FOL-R_545~50PKM58.583unfavorable

[0095] In Table 2 above, the ion scores are -10*Log(p), where p<0.05 indicates identity or extensive homology. (NP: not prognostic; unfavorable: high expression is not desirable in the given cancer.) In Table 2 above, individual ion scores (for Mascot score histogram) of peptides detected via LC-MS / MS with ion scores higher than 80 were selected as candidates that were statistically significantly enriched in the FOLFIRINOX-resistant group. The band labels indicate from which band each candidate was excised, and the molecular weight (MW) of the proteins in the gel, which is the native mass of the protein, is listed in the table that follows. Whether high expression of the selected candidates is prognostic in pancreatic adenocarcinoma is also indicated.

[0096]

[0097] Information on 554 candidate proteins from the Human Protein Atlas (https: / www.proteinatlas.org / ). Gene name RAB10 IARS2 KRT8 KRT19 PKMTissue (cell type) Pancreas (Exocrine glandular cells) Medium Medium High High Low Pancreas (Pancreatic endocrine cells) High High ND ND Medium Cholangiocytes (Cholangiocytes) Low Low High High Medium Liver (Hepatocytes) Medium Medium Medium ND ND Endometrium (Glandular cells) Medium High High High Medium Prognostic marker Pancreatic cancer unfavorable unfavorable unfavorable unfavorable unfavorable Liver cancer unfavorable NP NP NP unfavorable Endometrial cancer unfavorable NP NP NP NP Subcellular Predicted location Intracellular Intracellular Intracellular Intracellular Amino acid 2001, 262483400531

[0098]

[0099] Summary of bulk RNA-seq and plasma membrane (PM) proteome results (experimental data obtained from the AsPC-1 pancreatic cancer cell line)Gene name (protein name) RAB10IARS2KRT8KRT19PKM mRNA level (RNA-seq, %) Parent 100 100 100 100 100 Folfirinox-resistance 95.15 109.87 91.33 100.54 105.46 PM enrichment (LC-MS / MS) Parent NDNDNDNDND Folfirinox-resistance + unique + unique + unique + unique + unique PM fraction (LC-MS / MS) Fraction 1 (150 kDa ~) Fraction 2 (90~150KDa) Fraction 5 (45~50KDa) Fraction 5 (45~50KDa) Fraction 5 (45~50KDa) Predicted Protein size 23kDa 114 kDa 50kDa 44 kDa 58 kDa

[0100] Gene ontology-based functional clustering and fold analysis were performed on 24 candidates enriched in the R group with unbiased analysis (Fig. 1i and j). The subcellular localization of 25 candidates, including KRT8, KRT19, PKM, RAB10, and IARS, is predicted to be intracellular in the normal state, but was specifically detected at the plasma membrane of FOLFIRINOX-resistant pancreatic cancer cells (Fig. 1h).

[0101] Interestingly, although the native protein mass of RAB10 is 23 kDa, peptides were excised from SDS-PAGE slices containing proteins within the 200–400 kDa region (band designated FOL-R_1, Table 2) in plasma membrane protein LC-MS / MS analysis. MYH9, with a native protein mass of 228 kDa, was detected in the same slice as RAB10 (FOL-R_1, Table 2) and was identified as being retained at the plasma membrane by binding to RAB10. Furthermore, functional annotation confirmed that RAB10 is a unique redundant protein among functional clusters including transport, protein domain-specific binding, focal adhesion, cytoskeleton, and cytoplasm (Fig. 1j). These results suggest that RAB10 may serve as an essential regulator of FOLFIRINOX resistance at the plasma membrane of pancreatic cancer cells.

[0102]

[0103] 2-2. Confirmation of control of porfirinox-resistant pancreatic cancer due to RAB10 retention in the plasma membrane.

[0104] Next, we investigated the roles of RAB10 and MYH9 in FOLFIRINOX resistance in pancreatic cancer. Cell surface expression of RAB10, including MYH9, was enhanced 10.8-fold and 7.7-fold in the R group compared to the P group, respectively, but overall expression of the protein was not (Fig. 2a and b). However, cell surface expression levels of the PM marker protein Na,K-ATPase remained constant in both conditions, confirming the specificity of RAB10 and MYH9 as PM-enriched proteins associated with FOLFIRINOX resistance.

[0105] Furthermore, analysis of RAB10 gene knockdown by antisense oligonucleotides (ASOs) revealed that silencing RAB10 restored the chemosensitivity of pancreatic cancer cells to FOLFIRINOX (Fig. 2e-f). mRNA quantification confirmed the silencing efficiency of ASOs targeting the 5'-, 3'-, and internal (-In) regions of human RAB10 mRNA (Fig. 2d).

[0106] Parental and FOLFIRINOX-resistant AsPC-1 cells were treated with 10-fold serially diluted concentrations of FOLFIRINOX 48 hours after transfection with scrambled or RAB10-targeting ASOs. Cell viability was measured after 72 hours by WST-8 cytotoxicity assay. The IC50 of FOLFIRINOX-resistant AsPC-1 cells was reduced from 3.8-fold to 1.1-fold compared to parental cells, similar to chemosensitive PDAC cells (Fig. 2f).

[0107] Considering that the C subset of the ATP-binding cassette ABC transporter superfamily (ABCC), also known as multidrug resistance proteins (MRPs), is a transmembrane protein that interferes with drug sensitivity and is an essential ATP-dependent efflux transporter, we examined the mRNA expression levels of ABCCs in the P and R groups. The mRNA expression of ABCC1 (MRP1), a component contributing to multidrug resistance (MDR), including ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, ABCC6, ABCC10, and ABCC11 in the P and R groups, was statistically significantly increased in the R group (Fig. 7a, b). Therefore, we examined the cell surface expression of MRP1 and confirmed a 1.8-fold increase in expression in the R group (Fig. 2a, b). This supports the notion that MRP1 is retained on the plasma membrane instead of being degraded due to the disruption of RAB10-mediated endocytosis. Therefore, expression of RAB10 on the cell surface is important for promoting PDAC cells resistant to porfirinox.

[0108]

[0109] 2-3. Confirmation of the role of MYH9 as a motor protein that induces RAB10-mediated plasma membrane accumulation of MRP1.

[0110] In this example, the relationship between RAB10 and MYH9 was investigated using co-immunoprecipitation and antibody-based affinity capture mass spectrometry analysis to determine the underlying mechanism of MYH9 and RAB10 in regulating pancreatic cancer cells resistant to FOLFIRINOX.

[0111] Cells were harvested, homogenized to isolate the membrane fraction, pre-cleared with normal IgG to reduce nonspecific binding, and immunocaptured with RAB10 antibody. Anti-RAB10 captured protein complexes were then trypsin-digested and subjected to LC-MS / MS to compare the binding partners of RAB10 in parental and FOLFIRINOX-resistant PDAC (Fig. 3a and b). Results revealed that a total of 151 proteins were identified in the parental group (P) (25 proteins) and the FOLFIRINOX-resistant group (R) (126 proteins), of which 40 proteins were uniquely enriched in the R group (Fig. 3c). Therefore, 40 protein peptides were selected as potential binding partners of RAB10, among which individual ion scores were higher than 80.

[0112] Next, four candidates, including KRT19, KRT8, PKM, and MYH9 (Fig. 3c and Table 5), overlapped with the plasma membrane-enriched candidates selected in the primary LC-MS / MS analysis of Fig. 1 . Similarly, MYH9 (228 kDa) was a unique RAB10-associated protein detected in the 200–400 kDa protein range (FOL-R_1, Table 5 ), where RAB10 was observed in the plasma membrane of FOLFIRNOX-resistant PDAC.

[0113] Gene ontology-based functional clustering and fold enrichment analysis were performed on 24 candidates enriched in the R group in an unbiased analysis. In the FOLFIRINOX resistance setting, MYH9 was identified as a specific overlapping protein among functional clusters including stress fibers, actin filament binding, focal adhesion, and cytoskeletal organization summarized in the gene ontology analysis clustering of 40 cryptic RAB10 binding partners (Fig. 3e).

[0114]

[0115] RAB10-specific binding partners in FOLFIRINOX 566-resistant PDAC obtained by affinity capture mass spectrometry.번호밴드라벨유전자 명칭질량(kDa)Mascot ScorePDAC 예후FOLRINOX-R PM LC-MS / MS1FOL-R_5TUBB150.12763NPNA2FOL-R_6ACTB41.32011NPNA3FOL-R_5TUBB2A50.21666NPNA4FOL-R_5TUBA3C46.71341NPNA5FOL-R_4KRT1944.1945Unfavorableoverlap6FOL-R_5TUBB345.9891NPNA7FOL-R_5KRT1848.0772UnfavorableNA8FOL-R_5KRT1057.3644NPNA9FOL-R_6HSP90AB140.2600NPNA10FOL-R_5KRT858.4461Unfavorableoverlap11FOL-R_5PKM58.4398Unfavorableoverlap12FOL-R_1MYH9227.6397NPoverlap13FOL-R_5TUBA4A50.5365NPNA14FOL-R_5KRT853.7357NPNA15FOL-R_5TUBA3C46.8320NPNA16FOL-R_5PLEC52.0311NPNA17FOL-R_7TRAP122.7290NPNA18FOL-R_5EEF1G50.4288NPNA19FOL-R_1FLNB280.1264UnfavorableNA20FOL-R_3EEF296.2234NPNA21FOL-R_7SPTBN126.5221NPNA22FOL-R_4DDX1773.9204NPNA23FOL-R_3ACTN1105.6186NPNA24FOL-R_3KPNB198.4183NPNA25FOL-R_4KRT265.6176NPNA26FOL-R_6LDHA36.9170unfavorableNA27FOL-R_1FILIP1282.5150NPNA28FOL-R_4KRT166.2147NPNA29FOL-R_5KRT6B60.3146NPNA30FOL-R_6ACTBL242.3143NPNA31FOL-R_6SLC25A340.5139NPNA32FOL-R_6VAT141.6138NPNA33FOL-R_5TUFM49.8130NPNA34FOL-R_4KRT7762.1127NPNA35FOL-R_4HSPA1L77.9117NPNA36FOL-R_7CSF2RB11.7114NPNA37FOL-R_7YWHAZ35.599unfavorableNA38FOL-R_2VCL11796NPNA39FOL-R_2MYO1C122.587NPNA40FOL-R_3MVP99.682NPNA.

[0116] In Table 5 above, 40 of the 151 proteins are selected as confidential candidates with individual ion scores higher than 80. Whether high expression of the selected candidates is prognostic in pancreatic adenocarcinoma is also indicated. Proteins overlapping with the plasma membrane proteome candidates selected in Figure 1 are also annotated.

[0117] Myosin superfamily proteins, such as myosin II and myosin V, are known to be effectors of RAB protein-mediated vesicle trafficking along microfilaments. MYH9 (NMHC IIA) belongs to the nonmuscle myosin II family, consisting of two heavy chains and two light chains, and plays a central role in cell adhesion by cross-linking with actin. When protein samples were precipitated with antibodies against RAB10, protein-protein interactions between RAB10 and MYH9 were minimal under normal conditions. However, in FOLFIRINOX-resistant pancreatic cancer cells, the association between RAB10 and MYH9 increased to a 6.42-fold change (Fig. 3F and G), consistent with enhanced PM accumulation (Fig. 2A and B). Conversely, while RAB10-interacting myosin Vα binds cargo and promotes the movement of insulin-dependent GLUT4 vesicles to the PM, myosin II does not overlap with GLUT4 and remains localized to intracellular components in response to insulin stimulation. Therefore, these results imply a role for MYH9 as a key factor in RAB10-mediated vesicle trafficking in response to FOLFIRINOX treatment.

[0118]

[0119] [Sequence List]

[0120] Sequence number 1

[0121] scramble ASO

[0122] C*C*T*CTTACCTCAGTTACAATTT*A*T*A

[0123]

[0124] Sequence number 2

[0125] 5'-hRAB10 ASO

[0126] T*C*G*TACGTCTTCTTCGC*C*A*T

[0127]

[0128] Sequence number 3

[0129] 3'-hRAB10 ASO

[0130] T*C*A*GCAGCATTTGCTCT*T*C*C

[0131]

[0132] Sequence number 4

[0133] internal hRAB10 ASO

[0134] T*C*A*TCTATGTTTCTAAGCCATT*T*G*C

[0135]

[0136] Sequence number 5

[0137] GAPDH forward primer

[0138] 5'-AAT CCC ATC ACC ATC TTC CA-3'

[0139]

[0140] Sequence number 6

[0141] GAPDH reverse primer

[0142] 5'-TGG ACT CCA CGA CGT ACT CA-3'

[0143]

[0144] Sequence number 7

[0145] hRAB10 forward primer

[0146] 5'-CGA CCT GCT TTT CAA GCT GC-3'

[0147]

[0148] Sequence number 8

[0149] hRAB10 reverse primer

[0150] 5'-AGG CAT CAT CCG AAA AAC GA-3'

[0151]

[0152] Sequence number 9

[0153] hKRT8 forward primer

[0154] 5'-GGC CGT GGT TGT GAA GAA GA-3'

[0155]

[0156] Sequence number 10

[0157] hKRT8 reverse primer

[0158] 5'-CTT GGG CAG GAC GTC AGA G-3'

[0159]

[0160] Sequence number 11

[0161] hKRT19 forward primer

[0162] 5'-TGG AGA TGC AGA TCG AAG GC-3'

[0163]

[0164] Sequence number 12

[0165] hKRT19 reverse primer

[0166] 5'-GCC CCT CAG CGT ACT GAT TT-3'

[0167]

[0168] Sequence number 13

[0169] hPKM forward primer

[0170] 5'-TCA CGC TGG ATA ACG CCT AC-3'

[0171]

[0172] Sequence number 14

[0173] hPKM reverse primer

[0174] 5'-AGA TCT TGC TGC CCA CTT CC-3'

[0175]

[0176] Sequence number 15

[0177] hIARS2 forward primer

[0178] 5'-AGC CCA AGA GTG TTT TCC GT-3'

[0179]

[0180] Sequence number 16

[0181] hIARS2 reverse primer

[0182] 5'-TCT TCC AAC CCG GGC TTT TT-3'

[0183]

[0184] Sequence number 17

[0185] RAB10 protein

[0186] MAKKTYDLLFKLLLIGDSGVGKTCVLFRFSDDAFNTTFISTIGIDFKIKTVELQGKKIKLQIWDTAGQERFHTITTSYYRGAMMGIMLVYDITNGKSFENI SKWLRNIDEHANEDVERMLLGNKCDMDDKRVVPKGKGEQIAREHGIRFFETSAKANINIEKAFLTLAEDILRKTPVKEPNSENVDISSGGGGVTGWKSKCC

Claims

1. A biomarker composition for diagnosing therapeutic agent resistance of pancreatic cancer, comprising RAB10 or MYH9 protein.

2. In paragraph 1, A biomarker composition for diagnosing therapeutic agent resistance in pancreatic cancer patients, wherein the above RAB10 or MYH9 protein is detected in the cell plasma membrane.

3. In paragraph 1, A biomarker composition for diagnosing treatment resistance in pancreatic cancer patients, characterized in that the above treatment agent is porfirinox.

4. A kit for diagnosing pancreatic cancer treatment resistance, comprising an antibody or peptide that specifically binds to RAB10 protein, MYH9 protein or an immunogenic fragment thereof.

5. In paragraph 4, A kit for diagnosing pancreatic cancer treatment resistance, characterized in that the above treatment agent is porfirinox.

6. A composition for alleviating or treating resistance to a pancreatic cancer treatment agent, comprising an inhibitor of RAB10 protein expression.

7. In paragraph 6, A composition for alleviating or treating resistance to a therapeutic agent for pancreatic cancer patients, wherein the RAB10 protein expression inhibitor comprises an antisense oligonucleotide that specifically binds to the RAB10 protein.

8. In paragraph 6, A composition for alleviating or treating resistance to a pancreatic cancer treatment agent, characterized in that the above treatment agent is porfirinox.

9. A method for providing information for diagnosing resistance to pancreatic cancer treatment. (a) a step of obtaining a biological sample from a subject; (b) a step of measuring the expression level of RAB10 protein in the sample to obtain data on the expression level of RAB10 protein; (c) comparing the RAB10 protein expression level of the subject obtained in step (b) with the RAB10 protein expression level of the non-resistant group to the treatment; and (d) A method for providing information for diagnosing pancreatic cancer therapeutic agent resistance, comprising a step of determining that therapeutic agent resistance exists when the expression level of the RAB10 protein of the subject is higher than the expression level of the RAB10 protein of the non-resistant group.

10. In paragraph 9, A method for providing information for diagnosing treatment resistance in a pancreatic cancer patient, wherein the biological sample is pancreatic cancer tissue.

11. In paragraph 9, A method for providing information for diagnosing therapeutic agent resistance in a pancreatic cancer patient, wherein the step (d) determines that therapeutic agent resistance exists when the expression level of the RAB10 protein of the subject is 5 to 10 times higher than the expression level of the RAB10 protein of the non-resistant group.

Images