CN105214077B

CN105214077B - Application of USP33 in tumor

Info

Publication number: CN105214077B
Application number: CN201410242697.3A
Authority: CN
Inventors: 吴瑛
Original assignee: Zhejiang Askeli Biotechnology Co Ltd
Current assignee: Sichuan Asikeli Biotechnology Co ltd; Asclepius Suzhou Technology Company Group Co Ltd
Priority date: 2014-06-03
Filing date: 2014-06-03
Publication date: 2019-02-05
Anticipated expiration: 2034-06-03
Also published as: CN105214077A

Abstract

The application that the present invention relates to USP33 in tumour more particularly relates to USP33 as tumor prognosis and detects the application of marker and USP33 in tumour medicine exploitation.Data provided by the invention show that the decline of USP33 expression quantity is bad related to tumor prognosis in kinds of tumors patient；And USP33 albumen inhibits Nasopharyngeal neoplasms by regulating and controlling Slit-Robo signal path.Therefore, USP33 can be used as tumor prognosis detection marker, while USP33 can also be applied to the exploitation of tumour medicine, provide new method and scheme for the detection and treatment of tumour.

Description

Application of USP33 in tumor

Technical Field

The invention relates to the technical field of medicines, in particular to USP33 used as a tumor marker and application thereof in preparing a medicine for treating lung cancer.

Background

Lung cancer is the most common cancer, dying about 138 million people worldwide each year, with a 5-year survival rate of about 15%. Although there are many current treatments, including surgery, radiation, chemotherapy, and targeted therapies, and even combination therapies among them, the effects are not satisfactory. Lung cancer can be divided into small cell lung cancer SCLC and non-small cell lung cancer NSCLC. Non-small cell lung cancer including squamous cell carcinoma, adenocarcinoma, undifferentiated large cell carcinoma, adenosquamous carcinoma and bronchogenic carcinoma; small cell lung cancer, consisting of uniform small cells, is typically oat-like. NSCLC accounts for about 80% of cases. Current treatments for lung cancer are mainly based on tumor morphology and staging systems based on tumor lymph node metastasis TNM. A number of cancer suppressor genes have been discovered in lung cancer, including TP53, P16, LKB1/STK11, NF1, RASSF1, APC, BRG1, PTEN and RB. Various susceptibility genes have also been discovered recently, however, the endogenous mechanisms that inhibit the invasion and metastasis of lung cancer are still poorly understood.

Recent reports have shown that neuronal targeting molecules are thought to be involved in the invasion and metastasis processes of tumors. Slit gene was originally found in drosophila, its product is a family of secreted proteins that act as nerve-targeting molecules. Recent studies have shown that the slit gene is involved in the invasion and metastasis of different types of cancer cells. Slit proteins fulfill their function by binding to a single-transmembrane protein roundabout (robo). One of the proteins that regulate Slit-Robo signaling is ubiquitin-specific protease 33(USP 33). As one member of the ubiquitin-specific protease family, USP33 originally found a protein as a substrate bound to VHL E3 ligase. USP33 is believed to be essential in the Slit signaling pathway regulating midline site commissural axonal guidance.

In addition, the slit signal is involved in the function of inhibiting the migration of breast cancer cells, and USP33 also participates in the function, which indicates that USP33 may play an important role in cancer invasion and metastasis. However, the mechanism of how USP33 regulates slit signaling in lung cancer is unclear.

Disclosure of Invention

The invention aims to solve the technical problem of providing the application of USP33 in lung cancer, in particular the application of USP33 in preparing medicaments for detecting and treating tumors.

In order to solve the problems, the technical scheme provided by the first aspect of the invention is the application of USP33 in tumors, the application of USP33 as a tumor prognosis detection marker and the application of USP33 in tumor drug development.

Preferably, USP33 is used as a prognostic test marker for tumors including lung cancer, breast cancer, melanoma and acute myeloid leukemia.

Preferably, the use of USP33 for the preparation of a medicament for the treatment of tumours, said tumours being lung cancer.

Preferably, USP33 is targeted for tumor therapy.

Preferably, USP33 inhibits tumor cell metastasis by modulating the slit-Robo signaling pathway.

In a second aspect, the invention provides a diagnostic kit comprising a substance for detecting protein USP33, wherein the protein USP33 has the sequence Seq No. 1.

According to a third aspect of the present invention, there is provided a kit comprising a substance for detecting the USP33 gene, wherein the USP33 gene sequence is Seq No. 2.

In a fourth aspect, the invention provides the use of a diagnostic kit for detecting tumor and tumor prognosis USP33 expression.

The fifth aspect of the invention provides a tumor pharmaceutical composition, which contains the USP33 gene and/or the protein coded by the gene and pharmaceutically acceptable pharmaceutic adjuvants.

Preferably, the tumor is lung cancer.

In this study, the inventors demonstrated that inhibition of the migration process of lung cancer cells by Slit protein requires USP 33. USP33 modulates the levels of Robo receptors in lung cancer cells. This is a significant difference from the mode of action of USP33 on neurons and non-cancerous cells (Yuasa-Kawada et al, 2009a, b). In addition, USP33 plays a role as a tumor suppressor in lung cancer cells, since low expression of USP33 correlates strongly with a lower survival rate in lung cancer patients.

USP33 expression was significantly reduced in lung cancer tissues. High USP33 expression correlates with better prognosis. USP33 improved the stability of Robo1 and was necessary for the slit protein to inhibit tumor cell migration. These findings indicate that USP33 is a key participant in lung cancer development and is promising as a novel prognostic marker for lung cancer.

The USP33 provided by the invention can regulate the level of Robo receptors in lung cancer cells, improve the stability of Robo1 protein, and is necessary for the slit protein to inhibit the migration of tumor cells. Therefore, USP33 can be used as an important component of a medicament for treating tumors, particularly lung cancer, and is used together with other anti-cancer medicaments. In addition, USP33 can be used as a lung cancer prognosis detection marker, and provides a new method and scheme for detecting and treating tumors, particularly lung cancer.

Drawings

The invention is further described with reference to the following figures and examples:

figure 1 shows mRNA expression levels in 25 lung cancer and non-lung cancer tissue samples, with expression on the Y-axis and GAPDH gene as a reference, P < 0.001.

FIG. 2 immunohistochemical staining detects USP33 expression in lung cancer and control samples. Wherein, a and c are strong positive signals for detecting the expression of USP33 in normal tissues, b is an example of the positive expression of USP33 in adenocarcinoma, and d is an example of the positive expression of the USP33 in squamous cell carcinoma.

Figure 3 quantitative analysis of lung cancer samples (N-25) and control normal tissue samples (CTRL) (N-25) USP33 protein expression.

The calculation method of the USP 33-immunohistochemical score was as follows: USP33 immunohistochemistry score (percentage of positive tumor cells) x staining signal intensity. The USP33 protein expression was significantly lower in lung cancer than non-lung cancer tissue (. beta., P < 0.01).

FIG. 4 shows, from published data bases, a decrease in the expression of USP33 in a sample of lung cancer patients; wherein,

FIG. 4A shows the variation and expression of the USP33 gene in TCGA database lung cancer samples. Light blue indicates homozygous deletion; green indicates somatic mutation; pink indicates up-regulation of expression; blue indicates down-regulation of expression. The computational data on which these results depend comes from the cancer CBIO portal (, (http://www.oncomine.org).)

FIG. 4B shows the amino acid mutation of USP33 in TCGA database lung cancer samples.

FIG. 4C is a graph showing the analysis of the expression level of the USP33 gene of lung cancer patients in five groups of databases using Oncomine (http:// www.oncomine.org). In the five groups of databases, the expression level of USP33 is obviously reduced in the lung cancer sample compared with the non-tumor sample. LUAD: lung adenocarcinoma; LuSC: lung squamous cell carcinoma, Ctrl: and (6) comparison.

FIG. 5 shows the change of the expression of the USP33 gene in different tissue samples.

FIG. 6 shows that the low expression of the USP33 gene in different cancer databases means a shorter survival time; the details are as follows

Fig. 6A is data of the TCGA lung cancer database AgilentG4502A — 07 — 3, where n is 91 in USP33low and 91 in USP33 high; p is 0.0436, and P is 0.0436,

fig. 6B is the TCGA lung cancer database IlluminaHiSeq _ rnaseq v2 data, n 106 for USP33low and n 106 for USP33 high; p is equal to 0.0267,

FIG. 6C shows the data in the GSE3141 lung cancer database were analyzed by KM (http:// kmplot. com/analysis /). N is 28 in USP33low and 83 in USP33 high; p is 0.031 and P is 0.031,

FIG. 6D shows data in GSE31210 lung cancer database were analyzed by KM (http:// kmplot. com/analysis /). N is 113 in USP33low, and n is 113 in USP33 high; p is 0.034, and P is,

fig. 6E is the TCGA lung cancer database USP33low, USP33high, n 61; p is 0.0188 of the total weight of the composition,

fig. 6F shows the lung cancer database analyzed by KM for n 562 in USP33low and n 553 in USP33 high; p is 0.034, and P is,

fig. 6G is the TCGA melanoma database USP33low at n 145, USP33high at n 50; p is 0.0453, the ratio of P,

fig. 6H is the TCGA acute myeloid leukemia database, n 77 in USP33low and n 82 in USP33 high; p ═ 0.0282.

FIGS. 7A and 7B are photographs and a graph of a count analysis of H1299cells after treatment with control or slit proteins after transfection with siRNA or siUSP33, respectively, with no significant change in the number of cells in the control and slit-treated groups on the third and fourth days.

FIG. 8 shows that USP33 inhibits lung cancer cell metastasis by modulating slit through ubiquitination catalytic site; wherein, fig. 8A is a picture of H1299cells wound healing experiments transfected with control siRNA or siUSP 33; FIGS. 8B and 8E show cell migration distances. FIG. 8C shows that Western test demonstrated that siUSP33, but not control siRNA, inhibited the expression of USP33 gene in H1299 cells. Figure 8D is wild type USP33 or mutant USP33(C163A) used for placebo or slit controls. After the scratch, the cells of the edge will move to the right, the graph is taken at different time points after the scratch, and the white-point lines represent the 0 hour wound edge. FIG. 8F shows Western confirmation of the expression of wild-type and mutant genes (C163A) of USP33 in H1299 cells.

FIG.9 shows that USP33 interacts with Robo1 and affects the stability of Robo1 in lung cancer cells. Among them, FIG. 9A interaction of USP33 with Robo1 in H1299 cells. Co-immunoprecipitation experiments were performed in control and slit groups using control IgG or Robo1 antibody. Western blotting detection is carried out on the protein obtained by immunoprecipitation by using an antibody of USP33,

FIG. 9B shows a deubiquitination experiment performed on H1299 transfected Robo-HA, Flag-ubiquitin, Ctrl siRNA or siUSP 33. After MG132(20 mu mol/L) treatment for 6h, the HA antibody is used for carrying out co-immunoprecipitation experiment, and then Western blotting detection is carried out on the obtained protein,

FIG. 9C shows deubiquitination of transfected Robo-HA, wild-type USP33 or mutant USP33 and untransfected H1299 cells. After MG132(20 mu mol/L) treatment for 6h, the HA antibody is used for carrying out co-immunoprecipitation experiment, and then Western blotting detection is carried out on the obtained protein,

FIGS. 9D and 9E show expression of USP33 and Robo1 in H1299cells by Western blot after transfection of siRNAs USP33#1, USP33#2, control siRNAs,

FIG. 9F shows H1299cells transfected with siUSP33 or control siRNA and treated with CHX (50. mu.g/mL) for various periods of time to detect the total amount of Robo1 in the cells.

FIGS. 9G, 9H show Western blot assay after 1299cells were treated with MG132 (20. mu. mol/L) for 6H (no treatment as control) after transfection of siUSP33 or control siRNA.

Detailed Description

The following describes embodiments of the present invention with reference to the drawings.

Materials (I) and (II)

1. Laboratory animal or material sources and treatments

Lung cancer tissues and non-tumor lung tissues were from samples collected after pathology department diagnosis at the fourth university of military medical hospital, all with informed consent from patients and compliance with guidelines of the relevant agencies and countries. The lung cancer sample distribution is shown in table 1 below.

The tumor tissue samples removed by the operation are frozen and stored by liquid nitrogen rapidly for extracting RNA.

Lung cancer tissue samples were fixed overnight in 10% neutral formalin solution, paraffin-embedded sections, stained with hematoxylin and eosin according to standard protocols, and immunohistochemical analysis was performed.

TABLE 1 Lung cancer sample distribution

H1299cells (from ATCC cell bank), HEK293 cells (from ATCC cell bank), were cultured in 10% (v/v) fetal bovine serum, 50mg/ml penicillin/streptomycin DMEM medium at 37 ℃ and 5% carbon dioxide.

2. Drugs and reagents:

2.1 medicine

USP33 antibody was obtained from PROTEINTECH GROUP, Robo antibody was obtained from PROTEINTECH GROUP, and beta-actin antibody was obtained from PROTEINTECH GROUP; cycloheximide was purchased from Sigma; MG132 was purchased from Sigma. Trizol from Invitrogen

2.2 reagents

Lipofectamine2000 transfection reagent was purchased from Invitrogen; siRNAs from USP33 (#1:5'-UCUCGACAGUGGCUUAAUUAA-3',

#2:5'-GGAUUCAGUUGGUGAAAUUAC-3') and negative control siRNA (5'-CGUACGCGGAAUACUUCGATT-3') were synthesized by Shanghai Jima pharmaceutical technology, Inc.; SYBR green PCR mix was purchased from ThermoFisher Scientific,

2.3 instruments

Ultrospec5300pro spectrophotometer available from GE; the ScanScope XT scanner was purchased from Aperio, USA, and the real-time fluorescent quantitative PCR scanner was purchased from ABI.

The packet data is expressed by mean plus minus standard deviation. The T-test (two samples) or ANOVA (more than two samples) were subjected to statistical significance analysis. Survival analysis, analysis curves were plotted and analyzed using Prism software and log-rank. All experiments were repeated at least three times, with a 95% confidence level of p <0.05 of interest.

Second, experimental method and result analysis

1. Expression level of USP33 in lung cancer cells and non-tumor lung tissue cells.

1.1 analysis of USP33mRNA expression levels by quantitative RT-PCR method, USP33 expression was significantly reduced (P <0.001) (fig. 1) compared to non-tumor lung tissue samples.

Total RNA in lung cancer tissues was extracted using Trizol, the amount and purity of RNA were determined using an Ultrospec5300 spectrophotometer, genomic DNA was removed from RNA samples using DNase I, first strand cDNA was synthesized using oligo primer (dT) and reverse transcriptase (M-MLV RT) using 5. mu.g RNA as a template, and the following primers were used for amplification using a StepOne real-time quantitative PCR instrument from ABI:

USP33(5'-TGTGATGCTTAGGCAAGGAG-3',Seq No.3,

and 5'-GGCCCTCCACCATAAATAGA-3'), Seq No. 4;

Robo1(5'-GCATCGCTGGAAGTAGCCATACT-3',Seq No.5

and 5'-CTAGAAATGGTGGGCTCAGGAT-3') Seq No. 6;

GAPDH(5'-GGAGCGAGATCCCTCCAA AAT-3',Seq No.7

and 5'-GGCTGTTGTCATACTTCTCATGG-3'), Seq No. 8. And (3) amplification procedure:

at 95 deg.C, 15 seconds, 60 deg.C, 30 seconds, 72 deg.C, 30 seconds, for 40 cycles, utilize 2^-ΔΔCTThe method performs the calculation.

1.2 Next, the inventors examined USP33 protein expression in the samples by immunohistochemical staining (see FIG. 2 for specificity against USP 33).

Lung cancer tissue samples were immunochemical analyzed in 5 micron thick tumor sections fixed in 10% formalin and paraffin embedded. Dewaxed first, rehydrated and heated in a citric acid (pH 6.0) microwave oven for 20 minutes. Following antigen retrieval, endogenous peroxidase activity was blocked by treatment with 3% hydrogen peroxide for 10 minutes, followed by 30-minute obstructive incubation with goat serum. USP33 Primary antibody was incubated overnight at 4 ℃ on the slides followed by incubation of the biotin antibody for 30 minutes at room temperature. Finally, the sections were visualized by 3, 3-diaminobenzidine staining. Stained sections were digitized by Scanscope XT. The USP33 protein expression level is expressed by numerical values, and the average percentage of positive tumor cells is determined after at least five random areas are selected in each section and amplified by 200. USP33 intensity score was 0, negative; 1+, weak; 2+, mild 3+, severe. Positive tumor cells, the percentage of staining intensity was then scaled up to generate USP 33-immunohistochemical staining score.

In normal lung tissue, the USP33 staining signal showed a range of 73.33-300.00 (mean 198.18), while the staining signal varied from 0.00 to 266.00 (mean 142.38) in those lung cancer samples. USP33 expression levels in lung cancer samples were significantly lower than non-tumor lung tissue (P <0.01, fig. 2 and 3).

1.3 to investigate why changes in USP33 expression in lung cancer patients were due, the inventors analyzed 554 cases of data in the cBioPortal cancer genomic database (http:// cBioport. org). Survival and association of USP33 gene expression in lung cancer patients were analyzed using the TCGA cancer database and the online KM-Plotter database.

USP33mRNA was downregulated in 9 lung cancer cases, the gene was deleted in 2 lung cancer cases, and somatic mutations in the gene appeared in 9 additional cases (FIGS. 4A and 4B). Notably, the three USP33 mutations were located within the catalytic domain of USP33 (fig. 4B). Thus, about 4% of these lung cancer cases show homozygous deletion or somatic mutation or downregulation of mRNA expression levels. In addition, somatic USP33 missense mutations were found in samples of lung adenocarcinoma (4.3-6.2%) and lung squamous cell carcinoma (3.4%) (fig. 5). The dataset of the expression profile of USP33 in the Oncoine database containing 5 independent groups of lung cancer sample mRNA gene table data was further analyzed (http:// www.oncomine.org /). Notably, USP33mRNA expression levels were significantly reduced in lung cancer samples compared to control samples in all 5 sets of data (fig. 4C). Taken together, the downregulation of USP33 expression levels in these lung cancer samples was highly consistent.

2. Expression level and clinical results of USP33

To investigate the relationship between the expression level of USP33 and the clinic, the inventors analyzed the correlation between the expression level of USP33 and the survival time of patients by published microarray chip data. The inventors generated Kaplan-Meier (KM) survival curve data and an online KM-Plotter database from Cancer Genome Atlas (TCGA) data. For each set of data, lung cancer patients were divided into two groups according to the expression level of USP33 gene. Kaplan-Meier was used to analyze and evaluate the difference in survival time between the high expression USP33 and the low expression USP33 groups. In the database of all four lung cancers, higher USP33 expression levels were accompanied by longer survival of patients (fig. 6A-6D).

The inventors further analyzed the relationship of USP33 expression levels to survival in other types of cancer patients. The results show that in other types of cancer, including breast cancer (fig. 6E and 6F), melanoma (fig. 6G) and acute myeloid leukemia (AML, fig. 6H), short life cycles in a range were accompanied by low levels of expression of USP 33. These data indicate that USP33 plays a role as a tumor suppressor in a variety of human cancers.

3. USP33 participates in Slit signal mediated lung cancer cell metastasis inhibition

Previous researches of the inventor show that the expression level of slit protein in different tumor cells (including lung cancer and breast cancer) is obviously reduced, and slit plays an important role in preventing and treating further development of cancer. Also, the results of the existing studies have demonstrated that USP33 is involved in slit signaling in lung cancer. In order to explore a specific mechanism of action of USP33 in lung cancer cells, the inventors investigated how it could modulate the Slit signaling pathway to inhibit metastasis of lung cancer cells.

3.1 recombinant Slit2 production and wound healing assays

The Slit2 gene fused with the c-myc tag is transfected into HEK293 cells to produce a Slit2 full-length protein with the c-myc tag. Cells were cultured in DMEM medium with 5% fetal bovine serum. Slit2 protein was purified from the culture supernatant. Supernatants of untransfected HEK293 cells were used as controls.

In performing wound healing experiments, H1299cells were plated on collagen-coated photochemically etched grid slides, which were placed in 35mm petri dishes. Once the cells had accumulated, the wound area was created by carefully scraping a monolayer of cells with a sterile 10ul pipette tip. The wells were washed with PBS to remove detached cells, and then cultured in control and Slit 2-containing medium. Ten pictures were taken under an inverted microscope at 0 hours and 13-16 hours after wound formation. The level of cell migration was quantified by measuring the wound formed by the forward migration of cells from their initial position.

Cell wound healing experiments prove that the Slit protein can obviously reduce cell migration of H1299 lung cancer cells. This inhibition disappeared after USP33 was knocked out by specific siRNA (siUSP33) (fig. 8A-8C), indicating that USP33 affected the migration of lung cancer cells through the slit signaling pathway. It should also be noted that neither treatment of H1299cells with slit nor treatment of siUSP33 on H1299cells affected the proliferation or cell cycle of H1299cells in these experiments (see fig. 7).

To test whether the deubiquitinating activity of USP33 mediated this function, the inventors used the USP33 mutant for testing. The inventor constructs USP 33-C163A with a point mutation of a catalytic site for deubiquitination, transforms H1299cells by wild type (Wt) USP33 or mutant C163A-mutant USP33(Mt), and then cultures the cells by a culture medium containing Slit or a control culture medium without Slit, and can see that the mutation of USP33(C163A) can increase the migration of the H1299cells in the presence of Slit. The inventor can obtain that: the deubiquitinating active structural domain of the USP33 protein plays an important role in inhibiting the migration of lung cancer cells.

4. USP33 modulating stability of Robo1 protein

It has been reported that in breast cancer cells, USP33 acts to alter the intracellular distribution of Robo1 protein, i.e. to redistribute Robo1Robo1 from within the cell to the cell surface, without affecting the overall level of Robo1 protein. Then whether USP33 has similar function in lung cancer cells.

The inventors tested the interaction of USP33 with Robo1 using the co-immunoprecipitation method (fig. 9A), deletion of USP33 gene in H1299cells, and increased ubiquitination levels of Robo1Robo1 in the presence of MG-132 (inhibitor of the ubiquitin-proteasome system) (fig. 9B). Overexpression of the wild-type USP33 gene (instead of the catalytically inactive mutant C163A), the ubiquitination level of Robo1 was reduced (fig. 9C). Unexpectedly, the expression level of USP33 in H1299cells was down-regulated by the siRNA method, and the expression level of Robo1 protein was also reduced, contrary to the results in breast cancer cells (Fig.9D; Yuasa-Kawada et al, 2009 a). In contrast, overexpression of wild-type USP33, but not C163A mutated USP33, resulted in increased expression of Robo1 in H1299cells (fig. 9E).

To investigate how USP33 maintained the stability of Robo1 protein, the inventors performed the following experiments: h1299cells were transfected with control sirna (ctrl) or siUSP33, while cells were treated with cycloheximide (CHX, protein synthesis inhibitor) for various periods of time. The level of Robo1 protein expression in cell lysates was detected by Western Blot (WB) analysis. Robo1 level decreased 6 hours after cycloheximide treatment; the results of experiments in which Robo1 protein was almost completely degraded in siUSP33 transfected H1299cells compared to control siRNA 12 hours after treatment indicated that downregulation of USP33 expression levels reduced the half-life of Robo1 (FIG. 9F). The phenomenon of lowering of Robo1 protein levels by siUSP33 was inhibited by proteasome inhibitor MG-132 (FIG. 9G), indicating that Robo1 degradation in H1299cells is mainly achieved by the ubiquitin-proteasome system. These results indicate that USP33 can increase the stability of Robo1 protein and prevent its degradation by ubiquitin-proteasome in lung cancer cells.

Third, result analysis

The results of the inventors' studies provide evidence that USP33 is a new member in lung cancer, modulating the slit signaling pathway. The inventors demonstrated that the down-regulation of USP33 expression in lung cancer correlates with clinical outcome. USP33 inhibits lung cancer cell migration by modulating the activity of the slit signaling pathway. In addition, USP33 improves Robo1 protein stability by inhibiting protein degrading enzyme activity. These results reveal a previously unknown role of USP33 in mediating slit-robo signaling in lung cancer cells.

USP33 is involved in many cellular processes, the inventors' studies demonstrate for the first time that USP33 is a lung cancer-associated gene, and the amount of expression of this gene is reduced in lung cancer tissue samples, this down-regulated USP33 expression is in the lung cancer multi-microarray database, and is confirmed by real-time quantitative RT-PCR and immunohistochemical analysis, high expression of USP33 is well correlated with lung cancer patient prognosis, human USP33 gene is located in chromosome 1 p31.1, about 50% of the region alleles are absent in non-small cell lung cancer, in this region, some tumor suppressor genes have been identified, for example, with DnaJ-like heat shock protein (HLJ1) localized in this region that has been identified as inhibiting lung cancer cell proliferation, anchoring non-dependent growth, tumorigenesis, cell metastasis and invasion cAMP-dependent protein kinase β (PRKACB) is down-regulated in non-small cell lung cancer (NSCLC) tissues, and the up-regulation of PRKACB prevents the development of non-small cell lung cancer, the results of the present inventors indicate that the USP33 is a potential prognostic marker.

Recent studies have shown that changes in the expression of different genes in the ubiquitinated protein family are associated with different types of cancer, including lung cancer, among others. For example, USP44 is down-regulated in human lung adenocarcinoma. Lower levels of USP44 enzyme expression in patients indicate that overall survival will be significantly reduced. Rearrangement and deletion within this homozygote of deubiquitinating (BAP1) has been found in lung cancer cell lines. USP1/UAF1 may act as an oncogene because the inhibitor of USP1/UAF1 and cisplatin act synergistically in inhibiting the proliferation of non-small cell lung cancer cisplatin-resistant cells. The patent application research of the invention shows that: USP33 is a novel cancer suppressor gene in lung cancer.

In human lung cancer, low expression of Slit2 protein correlates with late stage lung cancer and poor prognosis of patients. Robo1 deficient mice exhibit bronchial epithelial hyperplasia and focal hyperplasia, a pathological hallmark associated with early stage lung cancer. In the research of the inventor, the Slit2 protein obviously inhibits the migration of lung cancer cells, and supports that a Slit-Robo signal channel has an inhibiting effect on lung cancer.

The inventors' studies demonstrated that the inhibition of breast cancer cell migration by slit is dependent on the action of USP33, in lung cancer cells USP33 interacts with Robo1 and is necessary for slit to inhibit migration of lung cancer cells. Ubiquitin-mediated modification plays a key role in regulating protein stability (Ciechanover and Schwartz, 1994). However, USP33 interacts with Robo1, which is required for slit-mediated redistribution of Robo1 to the plasma membrane, but does not affect the level of total Robo1 protein.

In this study, USP33 stabilized the expression level of Robo1 protein in lung cancer cells by inhibiting the ubiquitin proteasome pathway. USP33 regulates the transduction of slit signals in lung cancer cells, which requires USP33 enzyme activity, since after USP33 catalytic site mutation (C163A), slit no longer inhibits lung cancer cell migration. Interestingly, 4 catalytic domain missense mutations in USP33 have been identified in the cbioport lung cancer sample genomic database. A combination of the invention patent application studies for breast cancer could indicate that USP33 might modulate slit signaling by different mechanisms in different cancer cells.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited by the foregoing examples, which are provided to illustrate the principles of the invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention, which is also intended to be covered by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. The application of USP33 in the preparation of a drug for detecting the prognosis and development of tumors, wherein the tumors are lung cancer, melanoma and acute myeloid leukemia, and the prognosis refers to the life cycle of a patient.

2 . The application according to claim 1 , wherein the USP33 is used as a tumor suppressor gene. 3 .

The application according to claim 2, wherein in lung cancer, the USP33 acts as a tumor suppressor gene by regulating the slit-Robo signaling pathway.