Analysis method for classifying non-small cell lung cancer patients indicating prognosis of responsiveness to cisplatin

Abstract

The present invention provides an analysis method comprising a step for measuring the expression level of a gene, encoding at least one protease selected from the group consisting of USP36, USP37, USP47, USP49, and OTUD6B, in tumor cell samples isolated in vitro from non-small cell lung cancer patients, the expression level being measured in order to provide information that indicates the prognosis of the responsiveness to cisplatin and is necessary for classifying non-small cell lung cancer patients. In addition, the present invention provides a kit that is for classifying non-small cell lung cancer patients and indicates the prognosis of the responsiveness to cisplatin, the kit comprising a molecule capable of measuring the expression level of a gene encoding the protein.

Description

An analytical method for classifying non-small cell lung cancer patients according to their prognostic response to cisplatin.

[0001] The present invention relates to an analytical method and kit for classifying non-small cell lung cancer patients with a prognostic response to cisplatin. More specifically, the present invention relates to an analytical method for providing information necessary for classifying non-small cell lung cancer patients with a prognostic response to cisplatin, comprising a step of measuring the expression level of a gene encoding a specific proteolytic enzyme, and a kit used in the analytical method.

[0002] Cancer was ranked as the leading cause of death in Korea in 2021, and lung cancer is one of the most common cancers worldwide. Furthermore, according to the National Cancer Center's 2020 cancer registration statistics, lung cancer has the highest mortality rate among major cancers, with a five-year survival rate of only 36.8%. Its incidence rate is 11.7% of all cancers, a difference of 0.1 percentage points, placing it second.

[0003] Compared to thyroid cancer, which has a 100% five-year survival rate, lung cancer is one of the most lethal cancers. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers. Chemotherapy is primarily used for patients with stage III or later NSCLC to delay metastasis and alleviate symptoms. It is also used before or after surgery for some patients with stages I and II.

[0004] Cisplatin is a widely used treatment for non-small cell lung cancer (NSCLC) and was the first platinum-based therapy approved by the US FDA. However, despite its promising initial response rate, most patients receiving cisplatin therapy develop resistance. Cisplatin resistance reduces treatment efficacy and is a contributing factor to tumor proliferation. Therefore, early identification of patients with NSCLC at risk for cisplatin response plays a crucial role in determining treatment strategies. However, effective prognostic biomarkers for identifying cisplatin-responsive patients in NSCLC have not been reported.

[0005] To identify biomarkers that can determine cisplatin responsiveness in non-small cell lung cancer (NSCLC), we analyzed protease genes that exhibited differential expression between NSCLC cells and cisplatin-treated NSCLC cells using multiplex polymerase chain reaction (RT-PCR). As a result, we discovered that genes encoding five previously unknown proteases associated with cisplatin responsiveness showed significant differences in expression in cisplatin-treated NSCLC, and we validated these results using quantitative real-time PCR (qRT-PCR). Therefore, these genes encoding proteases can be used to analyze cisplatin responsiveness at the molecular level, and thus, they can be useful as biomarkers for assessing the prognosis of cisplatin responsiveness in NSCLC patients treated with cisplatin.

[0006] Accordingly, the present invention aims to provide an analysis method using a gene encoding a specific protein degradation regulatory enzyme to provide information necessary for classifying non-small cell lung cancer patients showing a prognosis of responsiveness to cisplatin.

[0007] In addition, the present invention aims to provide a kit for classifying non-small cell lung cancer patients indicating a prognosis of responsiveness to cisplatin, which comprises a molecule capable of measuring the expression level of a gene encoding the above-mentioned protein degradation regulatory enzyme.

[0008] According to one aspect of the present invention, in order to provide information necessary for classifying non-small cell lung cancer patients showing a prognosis of responsiveness to cisplatin, an analysis method is provided, which comprises the step of measuring the expression level of a gene encoding a proteolytic enzyme selected from the group consisting of USP36, USP37, USP47, USP49 and OTUD6B in a tumor cell sample isolated from a non-small cell lung cancer patient.

[0009] Measurement of the expression level of a gene encoding the above-mentioned protein degradation regulatory enzyme can be performed by measuring the amount of mRNA of the gene. For example, measurement of the amount of mRNA can be performed by reverse transcription polymerase chain reaction (RT-PCR) or quantitative real-time polymerase chain reaction (qRT-PCR).

[0010] In one embodiment of the analysis method of the present invention, if a gene encoding USP36, USP37, USP49, or OTUD6B is expressed at a low level in a tumor cell sample isolated from a non-small cell lung cancer patient administered cisplatin, the patient can be classified as a non-small cell lung cancer patient showing a prognosis of responsiveness to cisplatin. In the above embodiment, the measurement of the expression level of the gene can be performed by measuring the amount of mRNA using the primer set of SEQ ID NOs: 3 and 4, the primer set of SEQ ID NOs: 5 and 6, the primer set of SEQ ID NOs: 9 and 10, or the primer set of SEQ ID NOs: 11 and 12.

[0011] In another embodiment of the analysis method of the present invention, if a gene encoding USP47 is highly expressed in a tumor cell sample isolated from a non-small cell lung cancer patient administered cisplatin, the patient can be classified as a non-small cell lung cancer patient with a prognosis of responsiveness to cisplatin. In the above embodiment, the expression level of the gene can be measured by measuring the amount of mRNA using the primer sets of SEQ ID NOs: 7 and 8.

[0012] According to another aspect of the present invention, there is provided a kit for classifying non-small cell lung cancer patients showing a prognosis of responsiveness to cisplatin, the kit comprising a molecule capable of measuring the expression level of a gene encoding a proteolytic enzyme selected from the group consisting of USP36, USP37, USP47, USP49 and OTUD6B, wherein the molecule is a primer having a complementary sequence specific to the gene encoding the proteolytic enzyme.

[0013] In the kit of the present invention, the primer may have one or more base sequences selected from the group consisting of SEQ ID NOs: 3 to 12. In addition, the kit of the present invention may be in the form of a microarray in which the primer is immobilized on a substrate.

[0014] According to the present invention, it has been revealed that genes encoding specific proteolytic enzymes, namely, genes encoding USP36, USP37, USP47, USP49, or OTUD6B, show significant differences in expression in non-small cell lung cancer treated with cisplatin. Therefore, these genes can be used to analyze responsiveness to cisplatin at the molecular biological level, and thus can be usefully used as biomarkers for analyzing the prognosis of responsiveness to cisplatin in non-small cell lung cancer patients administered cisplatin. Therefore, the analysis method and kit according to the present invention using these genes as biomarkers can be usefully used for classifying non-small cell lung cancer patients showing a prognosis of responsiveness to cisplatin.

[0015] Figures 1a to 1e show the results of measuring the mRNA expression of decreased (Figures 1a and 1d) and increased (Figure 1e) proteolytic enzymes upon treatment with cisplatin in non-small cell lung cancer cell line A549 using multiplex polymerase chain reaction (A: A549 cells, B: A549 cells treated with cisplatin).

[0016] Figure 2 shows the results of statistical analysis of the relative mRNA expression ratios of decreased proteolytic enzymes (USP35, USP36, USP37, USP49, and OTUD6B) and increased proteolytic enzymes (USP47) in cisplatin-treated A549 cells compared to the non-small cell lung cancer cell line A549, using quantitative real-time polymerase chain reaction. (***: p < 0.001, **: 0.001 < p < 0.01, *: 0.01 < p < 0.05, ns: p > 0.05)

[0017] In this specification, “prognosis of responsiveness to cisplatin” means that when cisplatin is additionally administered to a non-small cell lung cancer patient who has been administered cisplatin, the non-small cell lung cancer or its symptoms are treated, alleviated, or relieved with a significantly lower or no induction of resistance.

[0018] As used herein, the term "tumor cell sample isolated from a non-small cell lung cancer patient" refers to a cell or tissue sample isolated from the tumor cells of a non-small cell lung cancer patient through a biopsy or the like. Hospitals typically collect non-small cell lung cancer tumor cells and tissue from patients and perform various tests to diagnose and establish treatment plans for non-small cell lung cancer patients. Therefore, as used herein, the term "tumor cell sample isolated from a non-small cell lung cancer patient" refers to a cell or tissue sample isolated from a patient for tissue examination or the like at a hospital.

[0019] The present inventors used a protein degradation regulatory enzyme gene primer set developed in this laboratory (including, for example, the primer set of Republic of Korea Patent Publication No. 10-2018-0050098) to identify protein degradation regulatory enzyme genes whose expression levels specifically differ in non-small cell lung cancer cells according to cisplatin treatment through multiplex polymerase chain reaction. As a result, when cisplatin was treated in the non-small cell lung cancer cell line A549, we discovered protein degradation regulatory enzyme genes that were specifically underexpressed or overexpressed, namely USP35, USP36, USP37, USP47, USP49, or OTUD6B, and additionally verified these genes by performing quantitative real-time polymerase chain reaction to quantify the differences in mRNA expression. The above multiplex polymerase chain reaction and quantitative real-time polymerase chain reaction analyses revealed that the mRNA expression of USP35, USP36, USP37, USP49, and OTUD6B was downregulated in cisplatin-treated non-small cell lung cancer cell lines, whereas the mRNA expression of USP47 was upregulated. In particular, USP36, USP37, USP47, USP49, and OTUD6B have not been previously known to be associated with cisplatin responsiveness. Therefore, the genes encoding USP36, USP37, USP47, USP49, or OTUD6B may be useful as biomarkers for analyzing the prognosis of cisplatin responsiveness in non-small cell lung cancer patients administered cisplatin.

[0020] Accordingly, the present invention provides an analysis method comprising a step of measuring the expression level of a gene encoding a proteolytic enzyme selected from the group consisting of USP36, USP37, USP47, USP49 and OTUD6B in a tumor cell sample isolated from a non-small cell lung cancer patient in order to provide information necessary for classification (including diagnosis or identification) of a non-small cell lung cancer patient showing a prognosis of responsiveness to cisplatin.

[0021] The protein sequences of the protein degradation regulatory enzymes USP36, USP37, USP47, USP49 and OTUD6B used as biomarkers in the analysis method of the present invention and the base sequences of the genes encoding them are all known, and therefore, the known protein and gene sequences can be used in the analysis method of the present invention. The NCBI accession number of the USP36 (ubiquitin-specific peptidase 36) protein is NP_001308220, NP_001372098, etc., and the NCBI accession number of the mRNA encoding it is NM_001321291, NM_001385169, etc. The NCBI accession number of the USP37 (ubiquitin-specific peptidase 37) protein is NP_065986, and the NCBI accession number of the mRNA encoding it is NM_020935. The NCBI accession numbers of the USP47 (ubiquitin-specific peptidase 47) protein are NP_001269588, XP_005253055, etc., and the NCBI accession numbers of the mRNA encoding it are NM_001282659, XM_005252998, etc. The NCBI accession numbers of USP49 (ubiquitin-specific peptidase 49) protein are NP_001273483, NP_001371471, NP_061031, etc., and the NCBI accession numbers of the mRNA encoding it are NM_001286554, NM_001384542, NM_018561, etc. The NCBI accession numbers of OTUD6B (ovarian tumor deubiquitinase 6B) protein are NP_001273674, NP_057107, etc., and the NCBI accession numbers of the mRNA encoding it are NM_001286745, NM_016023, etc.

[0022] The expression level of the gene encoding the above-mentioned protein degradation regulatory enzyme can be measured according to a method commonly used in the field of biotechnology. For example, the expression level of the gene encoding the above-mentioned protein degradation regulatory enzyme can be measured by measuring the amount of mRNA of the gene, and the measurement of the mRNA amount can be performed by a method such as reverse transcription-PCR (RT-PCR) or quantitative real-time PCR (qRT-PCR).

[0023] In one embodiment of the analysis method of the present invention, if a gene encoding USP36, USP37, USP49, or OTUD6B is expressed at a low level in a tumor cell sample isolated from a non-small cell lung cancer patient administered cisplatin, the patient can be classified as a non-small cell lung cancer patient showing a prognosis of responsiveness to cisplatin. In the above embodiment, the measurement of the expression level of the gene can be performed by measuring the amount of mRNA using the primer set of SEQ ID NOs: 3 and 4, the primer set of SEQ ID NOs: 5 and 6, the primer set of SEQ ID NOs: 9 and 10, or the primer set of SEQ ID NOs: 11 and 12.

[0024] For example, the expression levels of genes encoding USP36, USP37, USP49, or OTUD6B in tumor cell samples isolated from non-small cell lung cancer patients before and after administration of cisplatin were measured by qRT-PCR; 2 through qRT-PCR -ΔΔCtBased on the mRNA expression level analyzed by the method, if the expression level of the gene encoding USP36, USP37, USP49 or OTUD6B in a tumor cell sample isolated from a non-small cell lung cancer patient after administration of cisplatin is significantly (for example, by about 10% or more) lower than the expression level of the gene encoding USP36, USP37, USP49 or OTUD6B in a tumor cell sample isolated from a non-small cell lung cancer patient before administration of cisplatin, the non-small cell lung cancer patient can be classified (diagnosed or identified) as a non-small cell lung cancer patient showing a prognosis of response to cisplatin.

[0025] In another embodiment of the analysis method of the present invention, if a gene encoding USP47 is highly expressed in a tumor cell sample isolated from a non-small cell lung cancer patient administered cisplatin, the patient can be classified as a non-small cell lung cancer patient with a prognosis of responsiveness to cisplatin. In the above embodiment, the expression level of the gene can be measured by measuring the amount of mRNA using the primer sets of SEQ ID NOs: 7 and 8.

[0026] For example, the expression level of the gene encoding USP47 in tumor cell samples isolated from non-small cell lung cancer patients before and after administration of cisplatin was measured by qRT-PCR; 2 through qRT-PCR -ΔΔCt Based on the mRNA expression level analyzed by the method, if the expression level of the gene encoding USP47 in a tumor cell sample isolated from a non-small cell lung cancer patient after administration of cisplatin is expressed significantly (for example, about 1.5 times or more) higher than the expression level of the gene encoding USP47 in a tumor cell sample isolated from a non-small cell lung cancer patient before administration of cisplatin, the non-small cell lung cancer patient can be classified (diagnosed or identified) as a non-small cell lung cancer patient showing a prognosis of response to cisplatin.

[0027] The present invention also provides a kit for classifying (including diagnosing or identifying) non-small cell lung cancer patients showing a prognosis of responsiveness to cisplatin, comprising a molecule capable of measuring the expression level of a gene encoding a proteolytic enzyme selected from the group consisting of USP36, USP37, USP47, USP49 and OTUD6B, wherein the molecule is a primer having a complementary sequence specific to the gene encoding the proteolytic enzyme.

[0028] In the kit of the present invention, a primer having a complementary sequence specific to a gene encoding the protein degradation regulatory enzyme can be manufactured according to a method commonly used in the field of biotechnology, and a diagnostic kit including the primer can also be manufactured. For example, the primer can have one or more base sequences selected from the group consisting of SEQ ID NOs: 3 to 12. In addition, the diagnostic kit of the present invention can be in the form of a chip, such as a DNA chip or a protein chip, by having a microarray form in which the primer is immobilized on a substrate.

[0029] Hereinafter, the present invention will be described in more detail through examples. However, the following examples are intended to illustrate the present invention, and the present invention is not limited by these examples.

[0030] Example

[0031] 1. Test method

[0032] (1) Cultivation of non-small cell lung cancer cell lines

[0033] Non-small cell lung cancer cell line A549 (American Type Culture Collection, Manassas, VA, USA) was cultured in RPMI 1640 medium (31800-022, Gibco, Grand Island, NY, USA) containing 10% FBS and 1% Antibiotic-Antimycotic (15240062, Gibco, Grand Island, NY, USA) at 37°C in a 5% CO2 incubator. 24 h before cell harvest, the experimental group was treated with 60 μM cisplatin.

[0034] (2) RNA extraction and cDNA synthesis

[0035] Each cell was harvested from the culture medium grown in RPMI 1640 medium, and RNA was extracted from A549 cells using Trizol solution (15596018, Thermo Fisher Scientific, Waltham, MA, USA). After RNA quantification and electrophoresis confirmation, cDNA was synthesized using a cDNA synthesis kit (CMRTK002, Cosmogenetech, Seoul, Korea) at a concentration of 1 μg of RNA, and multiplex polymerase chain reaction and quantitative real-time polymerase chain reaction were performed.

[0036] (3) Multiplex polymerase chain reaction

[0037] Reverse transcription-polymerase chain reaction was performed using primers (SEQ ID NOs: 9 and 10) capable of amplifying the housekeeping gene GAPDH and a protein degradation regulatory enzyme gene primer set prepared in this laboratory (including the primer set of Korean Patent Publication No. 10-2018-0050098) for cDNA diluted to 250 ng each. Multiplex polymerase chain reaction was performed when the amount of amplified GAPDH was constant. Multiplex polymerase chain reaction was performed by adding 2X premix for multiplex polymerase chain reaction (SMP01-M25h, SolGent, Daejeon, Korea) and 12 groups of protein degradation regulatory enzyme primers for multiplex polymerase chain reaction to each cDNA. The primer sequences used are as shown in Tables 1 to 5. PCR conditions were as follows: denaturation step at 95°C for 20 seconds, binding step at 60°C for 40 seconds, and extension step at 72°C for 60 seconds, for a total of 40 cycles. The above multiplex polymerase chain reaction analysis was repeated three times.

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047] (4) Quantitative real-time polymerase chain reaction

[0048] Using cDNA diluted to 100 ng as a template, StepOne TMQuantitative real-time polymerase chain reaction was performed on a Real-Time PCR System (4376357, Thermo Fisher Scientific, Waltham, MA, USA). SYBR TM Quantitative real-time polymerase chain reaction was performed using Green PCR Master Mix (4309155, Thermo Fisher Scientific, Waltham, MA, USA) PCR as follows: 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 45 s, and melting at 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. Afterwards, mRNA expression of USP35, USP37, and USP47 against β-actin was measured at 2 -ΔΔCt The primer sets used were analyzed using the following method. Table 6 shows the primer sets used.

[0049]

[0050] (5) Confirmation and analysis of the derived results

[0051] Densitometric analysis was performed using Image J (National Institutes of Health, Bethesda, MD, USA), and turkey analysis was performed using GraphPad Prism version 9 (GraphPad Software, La Jolla, CA, USA). One-way analysis of variance (ANOVA) was performed to indicate significant differences.

[0052] 2. Test results

[0053] RNA was extracted from the non-small cell lung cancer cell line A549 and cisplatin-treated A549, cDNA was synthesized, and then multiplex polymerase chain reaction was performed and analyzed by gel electrophoresis (Fig. 1). Figures 1a to 1d show the results of multiplex polymerase chain reaction analysis indicating the proteolytic enzyme genes that showed decreased mRNA expression in cisplatin-treated A549 cells. Figure 1e shows the results of multiplex polymerase chain reaction analysis indicating the proteolytic enzyme genes that showed increased mRNA expression in cisplatin-treated A549 cells. From the results of Figures 1a to 1e, it can be confirmed that the proteolytic enzyme genes that showed changes in mRNA expression in A549 cells and cisplatin-treated A549 are USP35, USP36, USP37, USP47, USP49, and OTUD6B.

[0054] Quantitative real-time polymerase chain reaction was performed to confirm the changes in mRNA expression of USP35, USP36, USP37, USP47, USP49, and OTUD6B in cisplatin-treated cells, thereby validating the multiplex polymerase chain reaction results (Fig. 2). Fig. 2 shows the results of a statistical analysis of the relative mRNA expression ratios of the proteolytic enzyme genes USP35, USP36, USP37, USP47, USP49, and OTUD6B based on the quantitative real-time polymerase chain reaction results. From the results in Figure 2, it can be confirmed that in cells treated with cisplatin, the mRNA levels of USP35 decreased by 0.8867 times, USP36 by 0.6689 times, USP37 by 0.8857 times, USP49 by 0.8122 times, and OTUD6B by 0.6877 times, and the mRNA levels of USP47 increased by 1.897 times.

[0055] 3. Consideration

[0056] Cisplatin is a platinum-based anticancer drug used to treat various cancers. However, frequent use of cisplatin can lead to resistance, leading to relapse and poor prognosis. Efforts are underway to overcome cisplatin resistance, and assessing cisplatin responsiveness can facilitate effective drug selection for patients. The present inventors compared the expression of protease-regulating enzyme genes in non-small cell lung cancer cells treated with or without cisplatin using multiplex polymerase chain reaction and quantitative real-time polymerase chain reaction. We confirmed decreased expression of USP35, USP36, USP37, USP49, and OTUD6B genes and increased expression of USP47.

[0057] A recent study (Liu et al., Laboratory Investigation (2022) 102: 524-533) reported that random inhibition of USP35 gene expression increased cisplatin sensitivity in non-small cell lung cancer cells. These results support the experimental results of the present inventors, and in addition to USP35, a cisplatin responsiveness model can be constructed centered on USP36, USP37, USP47, USP49, and OTUD6B. In particular, by confirming responsiveness to cisplatin, it can contribute to shortening treatment time and suggesting optimal treatment strategies. In addition, a kit for predicting lung cancer anticancer drug responsiveness can be developed using proteolytic enzymes as cisplatin responsiveness biomarkers, and it can be used in future research on cisplatin resistance mechanisms.

Claims

1. An analysis method comprising a step of measuring the expression level of a gene encoding a protein degradation regulatory enzyme selected from the group consisting of USP36, USP37, USP47, USP49, and OTUD6B among tumor cell samples isolated from a non-small cell lung cancer patient in order to provide information necessary for classifying non-small cell lung cancer patients showing a prognosis of responsiveness to cisplatin.

2. An analysis method according to claim 1, characterized in that the measurement of the expression level of a gene encoding the protein degradation regulating enzyme is performed by measuring the mRNA level of the gene.

3. An analysis method according to claim 2, characterized in that the measurement of the amount of mRNA is performed by reverse transcription polymerase chain reaction (RT-PCR) or quantitative real-time polymerase chain reaction (qRT-PCR).

4. An analysis method characterized in that, in any one of claims 1 to 3, when a gene encoding USP36, USP37, USP49 or OTUD6B is expressed at a low level in a tumor cell sample isolated from a non-small cell lung cancer patient administered cisplatin, the patient is classified as a non-small cell lung cancer patient showing a prognosis of responsiveness to cisplatin.

5. An analysis method according to claim 4, characterized in that the measurement of the expression level of the gene is performed by measuring the amount of mRNA using a primer set of SEQ ID NOs: 3 and 4, a primer set of SEQ ID NOs: 5 and 6, a primer set of SEQ ID NOs: 9 and 10, or a primer set of SEQ ID NOs: 11 and 12.

6. An analysis method characterized in that, in any one of claims 1 to 3, if a gene encoding USP47 is highly expressed in a tumor cell sample isolated from a non-small cell lung cancer patient administered cisplatin, the patient is classified as a non-small cell lung cancer patient showing a prognosis of responsiveness to cisplatin.

7. An analysis method according to claim 6, characterized in that the measurement of the expression level of the gene is performed by measuring the amount of mRNA using a primer set of sequence numbers 7 and 8.

8. A kit for classifying non-small cell lung cancer patients showing a prognosis of responsiveness to cisplatin, comprising a molecule capable of measuring the expression level of a gene encoding a proteolytic enzyme selected from the group consisting of USP36, USP37, USP47, USP49 and OTUD6B, wherein the molecule is a primer having a complementary sequence specific to the gene encoding the proteolytic enzyme.

9. A kit according to claim 8, characterized in that the primer has at least one base sequence selected from the group consisting of sequence numbers 3 to 12.

10. A kit characterized in that the primer in the 8th paragraph is in the form of a microarray immobilized on a substrate.

Images