CN113499431A - Application of deubiquitinating enzyme USP28 in preparation of medicine for preventing or treating pancreatic cancer - Google Patents

Abstract

The invention discloses an application of deubiquitinase USP28 in preparing a medicament for preventing or treating pancreatic cancer, and researches show that USP28 expression in pancreatic cancer tumor tissues is higher than that of normal pancreatic tissues, and USP28 high expression is obviously related to malignant phenotype and shortened survival time of pancreatic cancer patients; overexpression of USP28 accelerates the growth of pancreatic cancer cells, while downregulation of USP28 inhibits the growth of pancreatic cancer cells in vitro and in vivo; USP28 promotes pancreatic cancer cell growth by accelerating cell cycle progression and inhibiting apoptosis. From the mechanism, USP28 de-ubiquitinates and stabilizes the transcription factor FOXM1, which is a key medium for Wnt/beta-catenin signaling, USP 28-mediated FOXM1 stabilization remarkably promotes the nuclear entry of beta-catenin, so that the Wnt/beta-catenin pathway is activated, and the restoration of FOXM1 expression can reduce the anti-tumor effect caused by USP28 down-regulation. The deubiquitinating enzyme USP28 promotes the pancreatic cancer development by enhancing the activation of the Wnt/beta-catenin signal channel mediated by FOXM1, and provides a powerful means for potential targeted treatment and prevention of pancreatic cancer patients in the future.

Description

Application of deubiquitinating enzyme USP28 in preparation of medicine for preventing or treating pancreatic cancer

Technical Field

The invention relates to the field of research on pathogenesis of pancreatic cancer, in particular to application of deubiquitinating enzyme USP28 in preparation of a medicine for preventing or treating pancreatic cancer, and more particularly relates to a mechanism for promoting pancreatic cancer growth by activating Wnt beta-catenin pathway through stabilizing FOXM1 of deubiquitinating enzyme USP28 and application of the mechanism in prevention or treatment of pancreatic cancer.

Background

Pancreatic Cancer (PC) is the seventh leading cause of cancer-related death worldwide. The poor prognosis is mainly manifested by a 5-year survival rate of about 8%, mainly due to late diagnosis and lack of effective treatment. Therefore, a better understanding of the molecular mechanisms involved in pancreatic cancer progression is an urgent need to identify new targets and develop new therapeutic strategies for treating pancreatic cancer.

Alterations in protein ubiquitination are often associated with tumors. DUBs counteract the activity of E3 ligase and are considered important molecules for the regulation of ubiquitination. Therefore, they have become potential therapeutic targets for tumors. USP28 is a member of the family of DUBs, involved in the physiological processes of cell proliferation, differentiation, apoptosis, DNA damage repair and stress response. Recent studies have shown that upregulation of USP28 expression is associated with poor clinical prognosis in a variety of cancers, such as colon, non-small cell lung and bladder cancer. Due to its deubiquitinase activity, USP28 interacts with Myc, catalyzing Myc deubiquitination, thereby promoting its stabilization and promoting tumor cell growth in colon and breast cancers. However, the biological function and expression pattern of USP28 in pancreatic cancer is not clear.

FOXM1 (forkhead box protein M1) acts as a key proliferation-related transcription factor that promotes tumor progression by activating expression of target genes at the transcriptional level. Elevated FOXM1 expression was observed in a variety of human cancers, and inhibition of FOXM1 significantly inhibited the malignant phenotype of cancer cells. Importantly, FOXM1 is a key regulator of cell cycle progression and several studies have shown that it also plays a key role in pancreatic cancer growth. Therefore, revealing the regulatory mechanism of FOXM1 will provide new insight into the pathogenesis of pancreatic cancer and new therapeutic strategies for this lethal tumor.

Disclosure of Invention

The invention aims to determine the effects of USP28 and FOXM1 in pancreatic cancer progression, and research the pancreatic cancer progression through USP28/FOXM 1/beta-catenin axis, so as to provide support for developing a biological targeted therapy and prevention drug for pancreatic cancer, and one of the technical problems to be solved by the invention is to provide an application of deubiquitinase USP28 in preparing a drug for preventing or treating pancreatic cancer.

The invention provides application of deubiquitinase USP28 in preparation of a medicine for preventing or treating pancreatic cancer.

The deubiquitinating enzyme USP28 activates Wnt beta-catenin pathway by stabilizing FOXM1 to promote pancreatic cancer growth.

The USP28 expression in the pancreatic cancer tumor tissue is higher than that in the normal pancreatic tissue, and the USP28 high expression is obviously related to the malignant phenotype and the shortened survival time of the pancreatic cancer patient.

Overexpression of USP28 accelerates the growth of pancreatic cancer cells, while downregulation of USP28 inhibits the growth of pancreatic cancer cells in vitro and in vivo.

USP28 promotes pancreatic cancer cell growth by promoting cell cycle progression and inhibiting apoptosis.

USP28 de-ubiquitinates and stabilizes FOXM1, which is a key mediator of Wnt/β -catenin signaling, USP 28-mediated FOXM1 stabilization significantly promotes nuclear β -catenin transactivation, which in turn leads to activation of Wnt/β -catenin pathway, and restoration of FOXM1 expression can alleviate anti-tumor effects caused by USP28 down-regulation.

This study determined that USP28 is a deubiquitinase for FOXM1 in pancreatic cancer. USP28 interacts with FOXM1 to reduce polyubiquitination of FOXM1, thereby stabilizing FOXM1, resulting in activation of the Wnt/β -catenin pathway. In addition, higher USP28 levels were strongly associated with progression and poor prognosis in pancreatic cancer patients. In addition, downregulation of USP28 expression inhibited the growth of pancreatic cancer cells in vitro and in vivo. These results indicate that USP28 is involved in the pathogenesis of pancreatic cancer by enhancing FOXM 1-mediated activation of the Wnt/β -catenin signaling pathway.

Advantageous effects

No matter in vivo or in vitro, USP28 plays an important role in the process of occurrence and development of pancreatic cancer cells, and provides a powerful means for potential targeted treatment and prevention of pancreatic cancer patients in the future according to the high expression result of USP28 in pancreatic cancer tissues.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1: in pancreatic cancer patients, overexpression of USP28 correlates with a poor prognosis.

A, representative H of USP28 in pancreatic cancer tissue and paired Normal tissue&E and IHC staining (magnification x 100, inset magnification x 400) scale bar, 50 μm. B. qRT-PCR analysis of USP28 mRNA expression in 102 pancreatic cancer tumors and paired normal tissues,^**P<0.01. c and D, protein immunoblotting was used to determine USP28 protein levels in pancreatic cancer tissues and paired normal tissues. GAPDH was used as loading control.^**P<0.01. E and F, Kaplan-Meier plot, represent the probability of progression free survival and overall survival for 102 pancreatic cancer patients based on the expression level of USP 28. Statistical analysis used student's t test and Log-Rank test.

FIG. 2: effect of USP28 on pancreatic cancer cell growth

A and B, mRNA and protein levels of USP28 in pancreatic cancer cell lines and human normal pancreatic ductal epithelial cells (H6c 7). C-F, CCK-8 experiments showed pancreatic cancer cell proliferation after over-expression (C and D) or down-regulation (E and F) of USP 28.^*P<0.05,^**P<0.01. Representative images (left) and quantitative analysis of pancreatic cancer cell colony formation by G-J, transfected USP28 overexpressing plasmid-P-USP 28(G and H) or interfering plasmid-shUSP 28(I and J)(Right).^*P<0.05,^**P<0.01. K, BxPC-3/shUSP28 cells were injected subcutaneously into nude mice and tumor volumes were measured at the indicated times; at the end of the experiment, tumors were dissected, photographed and weighed. n is 6, and n is 6,^*P<0.05,^**P<0.01. l, representative H from tumor tissue isolated from USP28 expression Down-regulated and control nude mice&And E, dyeing. USP28 expression down-regulated cell proliferation in nude mouse tumor tissue was detected by Ki67 staining. The proportion of ki 67-positive cells was counted, and the cell proliferation index (magnification × 100, insertion magnification × 400) was quantified.^**P<0.01. Scale bar, 50 μm.

FIG. 3: USP28 accelerates cell cycle progression and inhibits apoptosis

A-D, PC cell cycle was tested after overexpression or downregulation of USP28 expression, respectively. The results were expressed as a peak map (A, C), and cell distributions (B, D) at G0/G1 and S, G2/M phases were calculated.^*P<0.05. E and F, protein immunoblotting shows the expression of USP28, PCNA, cyclin D1 and CDK4 proteins in USP28 over-expressed AsPC-1 cells (E) or BxPC-3 cells (F) which down-regulate USP28 expression. GAPDH was used as loading control. The results are presented as scatter plots as apoptotic cells (G) and the percentage of Annexin-V positive cell population (H) in cells overexpressing USP28 was calculated.^*P<0.05,^**P<0.01. The results are presented as a scatter plot as the calculated percentage of apoptotic cells (I) and Annexin-V positive cell population (J) in cells with downregulation of USP28 expression.^*P<0.05. K and L, Western blotting to detect the expression of total protein and its active form of caspase 3 and PARP in USP28 overexpressing AspC-1 cells (K) or USP28 expression down-regulated BxPC-3 cells (L). GAPDH was used as loading control.

FIG. 4: USP28 activates the Wnt/beta-catenin signaling pathway in pancreatic cancer cells

(A) GSEA compared the gene set of Wnt targets in USP28 high expressing pancreatic cancer patients. Data were derived from the TCGA database. NES represents the normalized concentration score. B and C, phase of cells transfected with TOP-flash and FOP-flash vectors in USP28 over-expressed AsPC-1 cells (B) or USP28 expression down-regulated BxPC-3 cells (C) after treatment with 150ng/ml recombinant human Wnt3aAnd showing the luciferase activity level.^*P<0.05. D and E, detecting the total protein and nucleoprotein levels of beta-catenin in AsPC-1 cells (D) over-expressing USP28 or BxPC-3 cells (E) with USP28 expression down-regulated by Western blotting. GAPDH and Histone 3 were used as loading controls, respectively. F and G, IF analysis β -catenin nuclear levels in pancreatic cancer cells transduced with USP28 over-expressing plasmid-P-USP 28(G and H) or interfering plasmid-shUSP 28. Green signal is corresponding protein staining and blue signal is DAPI nuclear DNA staining. H and I, protein immunoblotting showed the expression of USP28, cyclin D1, c-Myc, VEGF and Survivin proteins in USP28 overexpressing AspC-1 cells (H) or in BxPC-3 cells (I) in which USP28 expression was down-regulated. GAPDH was used as loading control. Growth of USP28 overexpressing AsPC-1 cells by CCK8 technique (J) or colony formation assay (K) after treatment with J and K, β -catenin inhibitor (XAV-939).^*P<0.05。

FIG. 5: USP28 positively regulated FOXM1 protein levels in pancreatic cancer cells

A heat map of differentially expressed genes following downregulation of USP28 expression. B and C, Western blotting to detect the expression of USP28 and FOXM1 in USP28 over-expressed AsPC-1 cells (B) or USP28 expression down-regulated BxPC-3 cells (C). GAPDH was used as loading control. Total protein and nucleoprotein levels of β -catenin were determined by Western blotting of USP28 overexpressing AsPC-1 cells treated with FOXM1 inhibitor (RCM-1) (D), USP28 overexpressing PANC-1 cells treated with shFOXM1(E), and β -catenin. GAPDH and Histone 3 were used as loading controls, respectively. F and G, western immunoblotting to determine (F) and quantify (G) the levels of FOXM1 protein in pancreatic cancer tissue and paired normal tissue. GAPDH was used as loading control.^*P<0.05. H, scatter plots show that USP28 is positively correlated with FOXM1 at the pancreatic cancer protein level. Representative IHC staining (magnification x 100, insertion magnification x 400) of USP28 and FOXM1 in pancreatic cancer tissue and paired normal tissue. Scale bar, 50 μm.

FIG. 6: the efficiency of USP28 interference or overexpression in pancreatic cancer cells.

Protein expression levels of USP28 after transfection of shNC or shUSP28 in BxPC-3 and SW1990 cells were analyzed by Western blot A and B. GAPDH was used as loading control. C and D, protein immunoblotting to examine the protein expression level of USP28 after transfection of p-USP28 plasmid in AsPC-1 and PANC-1 cells. GAPDH was used as loading control.

FIG. 7: effect of overexpression of USP28 on pancreatic cancer cell growth.

A and B, AsPC-1/p-USP28 cells were injected subcutaneously into nude mice and tumor volumes were measured on the indicated days; and at the end of the experiment, tumors were dissected, photographed and weighed. n is 6, and n is 6,^*P<0.05，^**P<0.01。

FIG. 8: dual luciferase reporter gene assays overexpress USP28 or interfere with the activity of the luciferase reporter gene in USP28 pancreatic cancer cells.

A and B, interfering USP28/BxPC-3 cells (A) or over-expressing USP28/AsPC-1 cells (B) were transfected with TOP/FOP-Flash reporter plasmid, and reporter gene activity was detected by luciferase assay 48h after transfection. P < 0.05.

FIG. 9: immunofluorescence experiments examined the efficiency of USP28 interference or overexpression in pancreatic cancer cells.

A and B, pancreatic cancer cells transfected with p-USP28 or shUSP28 plasmid, and nuclear entry levels of USP28 were detected by immunofluorescence. The red signal represents staining of the corresponding protein and the blue signal represents staining of nuclear DNA by DAPI.

FIG. 10: treatment of over-expressing USP28AspC-1 cells with XAV-939 Western immunoblots showed protein expression profiles of USP28, β -catenin, c-Myc, and cyclin D1. GAPDH was used as loading control.

FIG. 11: western blot shows the expression of FOXM1 and β -catenin.

A and B, total and nucleoprotein levels of β -catenin in pancreatic cancer cells over-expressed in USP28 by Western blotting after treatment with RCM-1(A) or shFOXM1 (B). GAPDH and histone 3 were used as loading references, respectively.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Example 1

Materials and methods

Patient and tumor specimens

Matched pancreatic cancer tissue and normal pancreatic tissue specimens were obtained from 102 pancreatic cancer patients admitted to general surgery at the second affiliated hospital of Nanchang university in 2015-2019. The specimens taken off were immediately frozen in liquid nitrogen and stored at-80 ℃ for further analysis. All patients received written informed consent and the study procedure was approved by the ethical and research committee of the second subsidiary hospital, university of Nanchang. Table 1 summarizes the clinical characteristics of all patients.

Cell lines and cell cultures

Normal pancreatic ductal epithelial cell lines (H6C7 as a control) and pancreatic cancer cell lines (AsPC-1, PANC-1, BxPC-3, and SW1990) were purchased from the American type culture Collection (Manassas, Va.). The cell lines were cultured in medium (Gibco, USA) supplemented with 10% fetal bovine serum and 100 units/mL penicillin-streptomycin at 37 ℃ under 5% CO 2.

Quantitative real-time polymerase chain reaction

Total RNA was extracted from the cells using Trizol reagent (Invitrogen, USA). The real-time fluorescent quantitative assay was performed using SYBR Green kit (Clontech Laboratories, USA) and Applied

7900HT fast real-time fluorescence quantification apparatus (Thermo Fisher Scientific, USA). The primers used were as follows:

USP28：Forward 5'-ACTCAGACTATTGAACAGATGTACTGC-3'

Reverse 5'-CTGCATGCAAGCGATAAGG-3'；

FOXM1：Forward 5'-ACCGCTACTTGACATGGAC-3'

Reverse 5'-GGGAGGTTCGGTTTTGATGGTC-3'；

GAPDH：Forward 5'-CATACAGGAATGACTTGAC-3'

Reverse 5'-AACAGCGACACCCACTCCTC-3'

western blot, Immunohistochemistry (IHC) and Immunofluorescence (IF) analysis

For western blot analysis, equal amounts of cell lysates were separated by SDS/PAGE gel electrophoresis, electroporated onto PVDF (Millipore, USA) membranes, and blocked in 5% skim milk. Membranes were immunoblotted with the indicated primary antibody, followed by an appropriate horseradish peroxidase-conjugated anti-mouse/rabbit (KPL) or anti-goat (beijing cown Biotech, china) secondary antibody. The immunoreactive bands were developed by chemiluminescence using a chemiluminescence kit (Pierce, USA). Antibodies against USP28(1:1,000, abcam, ab126604), cyclin D1(1:1,000, Cell Signaling,55506), CDK4 (1:1,000, Cell Signaling,12790), PCNA (1:1,000, Cell Signaling,13110), cleared Caspase-3(1:1,000, abcam, ab2302) and Caspase-3(1:1,000) were used.

For IHC staining, matched cancerous and normal pancreatic tissue samples were fixed, embedded, sectioned and deparaffinized. Sections were then blocked with serum-free protein blocking buffer (DAKO, USA) for 30 min before being incubated with anti-USP 28(1:1, 000, abcam, ab126604), anti-FOXM 1(1:200, Cell Signaling, 5346), clear Caspase-3(1:200, abcam, ab2302) and anti-Ki 67(1:200, Cell Signaling, 2586). For IF analysis, after the indicated treatments, cells were fixed with 4% Paraformaldehyde (PFA) and incubated with 0.1% TritonX-100, followed by staining for β -catenin (1:100, abcam, ab32572), anti-USP 28(1:100, abcam, ab126604) and FOXM1(1:100, Cell Signaling, 5436). After incubation of the antibodies, nuclei were counterstained with DAPI. Then, the cells were observed with a laser scanning confocal microscope (Zeiss, Germany).

Overexpression, shRNA plasmid construction and cell transfection

Eukaryotic expression vectors encoding USP28 or FOXM1, as well as plasmids encoding short hairpin rna (shrna) against USP28 or FOXM1, were synthesized by GenePharma (shanghai, china). Pancreatic cancer cells were then transfected with these overexpression constructs or shRNA plasmids using Lipofectamine 3000(Invitrogen, USA) according to the manufacturer's instructions. Finally, G418 is used for screening and establishing pancreatic cancer cell lines stably transfected with a USP28 overexpression vector or a USP28 shRNA plasmid.

Cell proliferation and colony formation assays

For cell proliferation assays, pancreatic cancer cells were cultured in 96-well plates. At the indicated time points, viable cells were tested using the cell counting kit-8 (CCK-8) according to the manufacturer's instructions. For colony formation assays, pancreatic cancer cells transfected with either the over-expressed or shRNA plasmids were selected after 4-6 days in culture and then placed in 6-well plates (2000 cells per well). Finally, after 2-3 weeks of incubation, cells were incubated with fixation buffer (5% acetic acid and 5% methanol) and then stained with 0.5% crystal violet solution.

Flow cytometry analysis

Cell cycle experiments PC cells were harvested in exponential growth phase and single cell suspensions containing 1 x 105 cells were fixed in 70% ethanol. The cell cycle was then monitored using propidium iodide staining and detected using a FACScan flow cytometer (BD Biosciences, usa). For apoptosis detection, early and late apoptotic cells were assessed by annexin-V fluorescein isothiocyanate and propidium iodide apoptosis detection kit (BD Biosciences, usa) according to the manufacturer's instructions. Finally, stained cells were analyzed by FACScan flow cytometry.

Tumorigenicity assays and bioluminescence imaging

Firefly luciferase gene was stably transfected into injected pancreatic cancer cells and was able to be monitored regularly in vivo for tumor growth by bioluminescence imaging. For in vivo tumorigenicity testing, (1X 10)⁶100ml PBS) was injected subcutaneously into the ventral side of nude mice (male BALB/c-nu/nu, 6-8 weeks old). Then, for in vivo signal detection, mice were anesthetized with isoflurane and then imaged in a lumine series IVIS (in vivo imaging system) instrument (PerkinElmer, MA, USA). The animal work was approved by the ethical committee on animal experiments in the second subsidiary hospital of southern chang university.

Luciferase reporter Activity assay

Pancreatic cancer cells (1X 10 per well)⁵Individual cells) and TOPFlash luciferase reporter plasmid (for use inTo assess the transcriptional activity of β -catenin), pRL-CMV vector, and shUSP28 plasmid or USP28 vector co-transfected for overexpression studies using Lipofectamine 3000 transfection reagent. Cells were harvested 48 hours after transfection and luciferase activity was quantified using a dual luciferase reporter assay system (Promega, USA).

For exogenous Wnt3a treatment, recombinant human Wnt3a (R & D, 5036-WN) was dissolved in sterile PBS containing 0.1% FBS. Media containing 150ng/ml recombinant human Wnt3a was added to the cell cultures.

Liquid chromatography-mass spectrometry/mass spectrometry

LC-MS/MS analysis was performed as described previously. USP 28-silenced pancreatic cancer cells and control cells were analyzed by tandem mass spectrometry tag (TMT) labeling and LC-MS/MS. Whole cell lysates were extracted from these cells using lysis buffer (Sigma, USA) containing 1% protease inhibitor cocktail and quantified by the Aconitum mesaconii acid method. Equal amounts of protein (100. mu.g) from pancreatic cancer and control cells were reduced, alkylated, and then precipitated with acetone. The pellet was then resuspended in 200mM tetraethylammonium bromide and digested with trypsin. The samples obtained were then combined, separated by high performance liquid chromatography, and analyzed by LC-MS/MS.

Co-immunoprecipitation (Co-IP) and in vivo ubiquitination assays

For Co-IP analysis, cell lysates were incubated with specific primary antibody overnight at 4 ℃ and then with protein A/G-Sepharose beads (Santa Cruz, USA). The co-precipitated proteins were then collected and identified by immunoblot analysis for specific antibodies. For in vivo ubiquitination assays, cells were co-transfected with the aforementioned plasmids. After transfection, cells were treated with 50. mu.g/mL of the proteasome inhibitor MG132 for 12 h. Finally, cells were lysed for western blotting experiments and immunoprecipitation according to the same protocol as the Co-IP assay.

Statistical analysis

All data were expressed as standard error of mean average using GraphPad Prism 6(GraphPad software, usa). Significant differences were analyzed using student's t-test and two-tailed distribution. The survival curve is calculated by adopting a Kaplan-Meier method, and the significance is tested by adopting a logarithmic rank. P <0.05 was considered significant.

Results

USP28 is overexpressed in human pancreatic cancer specimens and is associated with poor prognosis

To investigate the role of USP28 in pancreatic cancer, we examined the expression of USP28 in 102 pancreatic cancer tissue specimens and their corresponding normal tissues. As shown in fig. 1A, Immunohistochemistry (IHC) results showed high expression of USP28 in 66.7% (68/102) pancreatic cancer tissue specimens. In addition, real-time fluorescence quantification experimental data show that the expression of USP28 mRNA is significantly increased in pancreatic cancer tissues compared to normal tissues. (FIG. 1B). Consistent with the increase in USP28 mRNA, USP28 protein levels were significantly increased in most cases (fig. 1C and D). These results suggest that USP28 is up-regulated in human pancreatic cancer tissues. We then evaluated the relationship between USP28 expression and clinical pathology factors in 102 pancreatic cancer patients (table 1). The results showed that the expression of USP28 had no significant correlation with age, histological type, presence or absence of lymph node metastasis; but significantly correlated with tumor size (P ═ 0.017), TNM staging (P <0.001), and degree of differentiation (P < 0.001). In addition, multivariate Cox regression analysis indicated that high expression of USP28 is an independent prognostic factor for poor survival in pancreatic cancer patients (table 2). To study the effectiveness of USP28 expression in predicting survival in pancreatic cancer patients, we analyzed the relationship between USP28 expression levels and survival in pancreatic cancer patients. As shown in FIGS. 1E and 1F, Kaplan-Meier analysis showed that the overall survival and disease-free survival of pancreatic cancer specimens with higher USP28 expression was significantly worse than pancreatic cancer specimens with lower USP28 expression. Taken together, these findings indicate that USP28 can serve as a valuable novel prognostic factor for human pancreatic cancer.

TABLE 1 relationship between the expression of deubiquitinase USP28 and clinical pathological characteristics

USP28 promotes pancreatic cancer cell growth in vitro and tumor progression in vivo

Real-time fluorescent quantitation and Western immunoblot analysis showed that USP28 was overexpressed in pancreatic cancer cell lines (AsPC-1, PANC-1, BxPC-3, and SW1990) compared to control H6C7 cells (FIGS. 2A and 2B). The frequent high expression of USP28 in pancreatic cancer tissues and cell lines prompted us to explore its carcinogenic role in pancreatic cancer. We constructed a USP28 overexpressing cell line using AsPC-1 and PANC-1 cells with relatively low endogenous USP28 expression (FIG. 6). Meanwhile, a USP28 expression stable down-regulation cell line is constructed by using a shRNA vector taking USP28as a target point and BxPC-3 and SW1990 cells with relatively high endogenous USP28 expression (figure 6). As demonstrated by the CCK-8 experiments, USP28 overexpressing AsPC-1 and PANC-1 cells significantly promoted cell proliferation (FIGS. 2C and 2D). In contrast, USP28 expression stably down-regulated BxPC-3 and SW1990 cell growth was inhibited (FIGS. 2E and 2F). Consistent with the findings from these experiments, plate clonogenic experiments showed that USP28 overexpression increased the cell proliferation potency of AsPC-1 and PANC-1 cells (fig. 2G and 2H), while USP28 down-regulation decreased the cell proliferation potency of BxPC-3 and SW1990 cells. (FIGS. 2I and J).

To assess the effect of USP28 on tumor growth in vivo, we performed subcutaneous neoplasia experiments in nude mice using USP28 expression stably down-regulated BxPC-3 cells. After 5 weeks of growth, a significant slowing of tumor volume and weight growth was observed (fig. 2K); this was accompanied by a decrease in cell proliferation (Ki67) (fig. 2L). In addition, USP28 overexpressing pancreatic cancer cells resulted in significantly faster tumor volumes and weights compared to their respective controls (fig. 7). Taken together, these data suggest that USP28 plays a key oncogenic role in promoting pancreatic cancer cell growth and tumorigenicity.

USP28 promotes pancreatic cancer cell growth by promoting cell cycle progression and inhibiting apoptosis

To investigate the mechanism of USP28 in promoting pancreatic cancer cell growth, we examined the effect of USP28 on cell cycle progression and apoptosis. Overexpression of USP28 in AsPC-1 cells resulted in a significant reduction in G0/G1 phase cells and an increase in S phase cells compared to controls (fig. 3A and 3B). In contrast, the down-regulation of USP28 expression in BxPC-3 cells had an opposite effect on cell cycle progression (fig. 3C and D). In addition, overexpression of USP28 enhanced the expression of proliferating cell nuclear antigen, cyclin D1 and cyclin-dependent kinase 4 (fig. 3E), while USP28 expression downregulated the expression of these key cell cycle regulators (fig. 3F). In addition, flow cytometry analysis showed that USP28 overexpression reduced early and late apoptosis in AsPC-1 cells (fig. 3G and H); however, downregulation of USP28 expression in BxPC-3 cells increased apoptosis induction (fig. 3I and J). The inhibitory effect of USP28 on apoptosis was further manifested by a decrease in caspase-3 and PARP cleavage type protein expression in USP28 overexpressing cells (figure 3K); however, the down-regulation of USP28 expression showed enhanced expression of these apoptotic factors (fig. 3L). Thus, these results indicate that USP28 promotes pancreatic cancer cell growth by promoting cell cycle progression and inhibiting apoptosis.

Wnt/beta-catenin is used as a downstream signal path of USP28 and is involved in the role of USP28 in pancreatic cancer cells

To explore the potential mechanisms of USP 28-mediated pancreatic carcinogenesis, we first performed a Gene Set Enrichment Analysis (GSEA) in the TCGA database to explore possible associations between USP28 and various signaling pathways. As shown in figure 4A, Hallmark _ Wnt _ Targets genome was significantly enriched in USP28 high expressing pancreatic cancer samples, suggesting that Wnt pathway is closely related to USP28 high expression in pancreatic cancer. Then, we tested the activation of the Wnt/β -catenin signaling pathway after changing USP28 expression, as shown in fig. 4B and C, we found that USP28 expression increase in AsPC-1 cells significantly improved the activity of TOP-Flash reporter after treatment with 150ng/ml recombinant human Wnt3 a; while the USP28 expression was down-regulated resulting in a decrease in the activity of the TOP-Flash reporter. Furthermore, the down-regulation of USP28 expression also inhibited the activation of the TOP-Flash reporter in BxPC-3 cells lacking recombinant human Wnt3a (figure 8). Importantly, nuclear β -catenin levels were significantly increased in pancreatic cancer cells overexpressing USP28 and decreased in cells downregulated by USP28 expression as compared to control cells (fig. 4D and 4E). However, changes in USP28 expression had no effect on the expression level of total β -catenin in pancreatic cancer cells (fig. 4D and 4E). Thus, changes in nuclear β -catenin expression in pancreatic cancer cells overexpressing USP28 and downregulating USP28 expression were further confirmed by immunofluorescence experiments (fig. 4F and 4G). In addition, immunofluorescence results showed that USP28 is expressed in pancreatic cancer cells overexpressing and downregulating USP28 expression (fig. 9). Overexpression of USP28 also increased the expression of Wnt/beta-catenin target genes, namely cyclind1, c-Myc, VEGF, survivin (FIG. 4H) are key genes related to cell proliferation and apoptosis. Conversely, downregulation of USP28 expression resulted in a decrease in expression of these genes (fig. 4I).

Next, we used the specific inhibitor of β -catenin, XAV-939, to inhibit the Wnt/β -catenin signaling pathway in USP28 overexpressing cells. Interestingly, XAV-939 significantly inhibited the enhancement of pancreatic cancer cell proliferation capacity caused by over-expression of USP28 (fig. 4J and 4K). Furthermore, our data show that increased expression of c-Myc and cyclin-D1 was significantly abolished by XAV-939 in pancreatic cancer cells overexpressing USP28 (figure 10). Taken together, these results indicate that the Wnt/β -catenin signaling pathway is critical for USP 28-mediated pancreatic cancer progression.

USP28 activates Wnt/beta-catenin signal channel by regulating expression of FOXM1

To determine the potential mechanism of USP28 to activate the Wnt/β -catenin signaling pathway in pancreatic cancer cells, we performed large-scale proteomics experiments based on LC-MS/MS analysis of TMT in pancreatic cancer cells with downregulation of USP28 expression. We found that FOXM1 protein was significantly down-regulated in pancreatic cancer cells with USP28 expression down-regulated (fig. 5A). Further studies found that FOXM1 protein levels were significantly elevated in USP28 overexpressing cells, but significantly reduced in USP28 expressing downregulated cells, indicating that USP28 positively regulates FOXM1 protein levels (figures 5B and 5C). Importantly, FOXM1 was reported to enhance β -catenin nuclear localization and transcriptional activity in pancreatic cancer cells. Therefore, we speculate that USP28 activates the Wnt/β -catenin signaling pathway by enhancing expression of FOXM1 in pancreatic cancer cells.

As expected, USP28 overexpression significantly increased nuclear β -catenin expression levels, while Robert Costa Memorial drug-1 (a small molecule inhibitor of FOXM 1) decreased nuclear β -catenin expression levels (FIG. 5D). FOXM1 silencing also inhibited USP28 overexpression of pancreatic cancer nuclear β -catenin expression levels (fig. 5E), indicating that FOXM1 may be involved in USP 28-mediated activation of Wnt/β -catenin signaling pathway. In addition, inhibition of FOXM1 reversed the increase in nuclear β -catenin strength in usp28 overexpressing PC cells (fig. 11).

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims (6)

1. Use of deubiquitinase USP28 in preparing medicine for preventing or treating pancreatic cancer is provided.

2. The use according to claim 1, wherein the deubiquitinase USP28 promotes pancreatic cancer growth by stabilizing FOXM1 activating the Wnt β -catenin pathway.

3. The use of claim 1, wherein USP28 expression is higher in pancreatic cancer tumor tissue than in normal pancreatic tissue, and USP28 high expression is significantly correlated with the malignant phenotype and shortened survival of pancreatic cancer patients.

4. The use of claim 1, wherein overexpression of USP28 accelerates the growth of pancreatic cancer cells and downregulation of USP28 inhibits the growth of pancreatic cancer cells in vitro and in vivo.

5. The use according to claim 1, wherein USP28 promotes pancreatic cancer cell growth by promoting cell cycle progression and inhibiting apoptosis.

6. The use according to claim 2, wherein USP28 de-ubiquitinylates and stabilizes FOXM1, which is a key mediator of Wnt/β -catenin signaling, and USP 28-mediated FOXM1 stabilization significantly promotes β -catenin internucleation, which in turn leads to activation of Wnt/β -catenin pathway, and restoration of FOXM1 expression can reduce anti-tumor effects caused by USP28 down-regulation.

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