Dig Dis Sci DOI 10.1007/s10620-017-4732-6

ORIGINAL ARTICLE

UBR5 Contributes to Colorectal Cancer Progression by Destabilizing the Tumor Suppressor ECRG4 Jin Wang1 • Xiaomu Zhao1 • Lan Jin1 • Guocong Wu1 • Yingchi Yang1

Received: 8 November 2016 / Accepted: 22 August 2017 Ó Springer Science+Business Media, LLC 2017

Abstract Background The E3 ligase UBR5 is aberrantly expressed in diverse types of cancer. However, its expression pattern and biological function in colorectal cancer (CRC) remain unclear. Methods We used RT-PCR, Western blot, and immunohistochemistry to measure UBR5 expression in CRC tissues and corresponding non-tumor tissues. The expression pattern of UBR5 in CRC tissues was determined by scoring system of immunohistochemical analysis and mRNA level by RT-PCR. The statistical analyses were applied to evaluate the associations of UBR5 expression with survival rate of patients. The UBR5 gene was overexpressed or silenced with lentiviral vectors in CRC cells. And, cell proliferation and apoptosis were measured using CCK8 assay and flow cytometry. Results We found that UBR5 is abundantly overexpressed in CRC tissues than adjacent non-cancerous tissues. We also found that high UBR5 level is positively correlated with progression and poor survival in CRC patients. In addition, further multivariate analysis indicated that UBR5 and TNM stage were independent prognostic factors for overall survival in patients with CRC. Furthermore, we demonstrated that the expression of UBR5 was significantly elevated in CRC cell lines. Overexpression of UBR5 enhanced in vitro cell proliferation and promoted in vivo tumor growth, whereas silencing UBR5 suppressed growth of CRC cells. Moreover, our findings show that UBR5

& Jin Wang [email protected] 1

Department of General Surgery, Beijing Friendship Hospital, Capital Medical University, 95 Yongan Road, Xicheng District, Beijing 100050, China

promotes CRC cell proliferation by inducing cell cycle progression and suppressing cell apoptosis. Finally, we found that UBR5 directly binds to the tumor suppressor esophageal cancer-related gene 4 (ECRG4) and increased its ubiquitination to reduce the protein stability of ECRG4. Conclusions We identified a tumorigenic role of UBR5 in CRC and provided a novel therapeutic target for CRC patients. Keywords UBR5  Colorectal cancer  Proliferation  Apoptosis  ECRG4

Introduction Colorectal cancer (CRC) is the third most common cancer worldwide and causes severe cancer-related mortalities [1]. Although much progress in treatment has been achieved for the past years [2], the prognosis of CRC remains unsatisfactory due to the high rate of recurrence and metastases [3]. To improve the prognosis of CRC patients, it is urgent to reveal the molecular mechanisms underlying CRC, which may contribute to the development of effective therapy for CRC. Ubiquitin protein ligase E3 component n-recognin 5 (UBR5), also known as EDD1, is an homologous to E6AP C-terminus (HECT) domain-containing ligase [4], which has been implicated in the regulation of metabolism, transcription, and apoptosis [5]. Previous studies indicate that UBR5 is linked to carcinogenesis, as aberrant elevation of UBR5 has been found in several cancers, including breast [6] and gastric cancer [7]. O’Brien et al. [8] have demonstrated that UBR5 can mediate therapeutic resistance in ovarian cancer through modulation of the DNA damage response. A recent research suggests that UBR5 is

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utilized by the HPV E6 oncogene to destabilize tumor suppressor TIP60 in cervical cancer [9]. Here we aimed to explore the biological role and functional mechanism of UBR5 in CRC. Esophageal cancer-related gene 4 (ECRG4) is a tumor suppressor gene, which was initially identified and cloned from normal human esophageal epithelium [10]. Recently, experiments have shown that ECRG4 was inversely correlated with cancer proliferation [11], invasiveness, metastasis, and progression [12]. More importantly, ECRG4 has been reported to serve as a novel tumor suppressor gene inhibiting CRC cell growth in vitro and in vivo [13]. However, the underlying mechanism by which ECRG4 is regulated in CRC is largely unknown. In the current study, our results revealed that UBR5 promotes the progression of CRC partially by regulating the protein stability of ECRG4. We found that UBR5 overexpression is associated with poor prognosis in CRC patients and enhances cell proliferation in vivo and in vitro. Furthermore, knockdown of UBR5 in CRC cells inhibits cell growth and induces cell apoptosis. Mechanistically, we found that UBR5 directly binds to ECGR4 and promotes ECGR4 ubiquitination to reduce the protein stability of ECRG4. Therefore, we identified that UBR5 as a novel regulator of CRC development and this may provide a potential therapeutic target for CRC patients.

Materials and Methods Tissue Samples Colonic carcinoma tissues and corresponding non-tumor tissues were collected from 80 patients who received curative surgery for CRC at the Department of General Surgery, Beijing Friendship Hospital, Capital Medical University. None of the patients received preoperative chemotherapy before recruitment to this study. The tissue samples were collected and used after obtaining approval from the Ethics Committee of Beijing Friendship Hospital, Capital Medical University. Informed consent was obtained from all of the patients who participated in this study according to committee’s regulations. Cell Lines and Cell Culture Normal human colon epithelial cell line (FHC) and four human colon cancer cell lines, including HT-29, HCT116, LOVO, and SW480, were purchased from American Type Culture Collection (Manassas, USA). FHC cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum. And, the CRC cell lines were routinely maintained in RPMI-1640 medium supplemented with

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10% fetal bovine serum at 37 °C in a humidified air atmosphere containing 5% CO2. Real-Time PCR (RT-PCR) Total RNA extraction and real-time RT-PCR were carried out using SYBR GreenER two-step kit (Invitrogen, Carlsbad, CA). The primer sequences were as follows: UBR5 (forward primer: 50 -ACGAGAAGGAAAGCACCATG-30 ; reverse primer: 50 -CTTCTCAGAAACTTCTCGTAAC-30 ), ECRG4 (forward primer: 50 -GGTTCTCCCTCGCAGCAC-30 ; reverse primer: 50 -TTGGAACAGGTGCTTCTCG-30 ), GAPDH (forward primer: 50 -CCTGCCGGTGACTAACCCTG-30 ; reverse primer: 50 -AGTTAAAAGCAGCCCTGGTG-30 ) were used as an internal control. The comparative 2-DCt method was applied to develop qRT-PCR method for analyzing the expression pattern of UBR5 in colorectal cancer. In present study, we considered that the DCt = -1 is the value of cut line between high and low expression of UBR5 in qRT-PCR. Western Blot Analysis A total of 40 mg protein was separated by 10% SDS-PAGE and then transferred to PVDF membrane. After blocking with 0.5% nonfat milk in TBST, the membranes were washed with PBS and then incubated with the primary antibody at 4 °C overnight. The antibodies used were as follows: anti-UBR5 antibody (1:1000, Abcam, USA), antiECRG4 (1:1000, Santa Cruz, USA), anti-ATMIN (1:1000, Santa Cruz, USA), anti-CDK9 (1:1000, Santa Cruz, USA), anti-Flag (1:1000, Santa Cruz, USA), anti-HA (Santa Cruz, USA), and anti-b-actin (1:1000, Abcam, USA). The signals were detected by enhanced chemiluminescence (Pierce, USA). Immunohistochemistry (IHC) Staining The clinical and tumor xenograft tissues were fixed, embedded, sectioned, and deparaffinized. IHC staining was performed using a Dako Envision System (Dako, USA) following the manufacturer’s protocol. Sections were blocked using serum-free protein block buffer (DAKO, CA, USA) for 30 min, after which they were incubated with anti-UBR5 (1:200, Abcam, USA), anti-C-caspase 3 (1:200, CST, USA) and anti-Ki-67 (1:200, Santa Cruz, USA). The pictures were recorded using a Nikon light microscope, and staining intensity analyzed using Nikon software (Nikon Inc., Melville, NY, USA). The pathological information was obtained from the detected CRC samples by using the German semiquantitative scoring method. Each specimen was scored for the intensity of nucleic, cytoplasmic, and membrane staining (no

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staining = 0; weak staining = 1, moderate staining = 2, strong staining = 3) and for the extent of stained cells (0% = 0, 1–24% = 1, 25–49% = 2, 50–74% = 3, 75–100% = 4). The final immunoreactive score was the product of the intensity score multiplied by the extent score. The value line of score between high and low expression of UBR is 4, a value [4 as a high score, and a value B4 as a low score. Constructs and Lentivirus Packaging Lentiviral vectors encoding the full-length UBR5 cDNA in pENTR221 vector purchased from GenePharma. Human UBR5 cDNA and ECRG4 cDNA were cloned to pcDNA3.1, and expressing tag was infused with the target genes. The pcDNA3.1-HA ubiquitin plasmid was obtained from Addgene. RNA Interference Sh-UBR5, sh-ECRG4, and control shRNA lentivirus particles were purchased from GenePharma. Lentiviral particles corresponding to the shRNA UBR5 (NM_001282873.1) or shRNA ECRG4 (NM_032411.2) target set were used, as well as a nontarget shRNA control. Specificity and efficacy of the UBR5 shRNA or ECRG4 shRNA were evaluated by Western blotting after transduction and puromycin selection in cells. Cell Counting Kit-8 Assay To measure cell proliferation, 5 9 103 cells/well were seeded in a 96-well plate and cultured in a 5% CO2 incubator at 37 °C. At the indicated time points, viable cells were counted using the Cell Counting Kit-8 (Dojindo Molecular Technology, USA). The OD value of the medium was detected using a spectrophotometer at 450-nm wavelength. Cell Cycle Analysis To examine cell cycle, 1 9 105 cells/well were seeded in a 6-well plate and cultured in a 5% CO2 incubator at 37 °C. After transfection for 48 h, the cells were fixed in 70% ethanol at -20 °C for 24 h. Then, the cells were analyzed by using a BD CycletestTM Plus DNA Reagent Kit (BD, USA) according to the manufacturer’s protocol. Apoptosis Assay After transfection for 48 h, cells were stained with Annexin V-FITC Apoptosis Detection Kits (BD, USA). In brief, cells were collected and washed with phosphate-buffered

saline and then resuspended in 19 binding buffer. Then 500 ll cell suspension, 5 ll Annexin V-FITC, and 5 ll propidium iodide solution were added into the test tube. After incubation for 15 min, cell apoptosis was analyzed using a FACS analyzer (BD, USA). Xenograft Nude Mouse Model The in vivo tumor xenograft experiments were performed with CRC cells as described previously [14]. Briefly, exponentially growing cells were implanted subcutaneously into the forelegs of 6-week-old male BALB/c athymic nu/nu mice (2 9 106 cells per animal). Tumors size was measured every 5 days by caliper to determine tumor volume using the formula: V = [length/ 2] 9 [width2]. All mice were killed 35 days after seeding of tumor cells, and the tumor weights measured. All animal experiments were approved by the Animal Care and Use Committee of Nanchang University. Statistical Analysis All values are expressed as the mean ± SEM of at least three experiment repeats. The Kaplan–Meier method was used to calculate the survival curve and log-rank test to determine statistical significance. Multivariate logistic regressions were used to assess the association between UBR5 expression and overall survival. The independent prognostic factors for overall survival were examined using the Cox proportional hazards models for the multivariate analysis, which were applied to estimate hazard ratios for mortality. The differences between groups were analyzed using two-tail Student’s t test and ANOVA. P values of less than 0.05 were considered statistically significant.

Results UBR5 Expression Is Upregulated in CRC Tissues and Is Closely Correlated with Survival in Patients with CRC To investigate the potential role of UBR5 in CRC development, we first examined the expression pattern of UBR5 in CRC tissue samples and the adjacent non-tumor tissues. We found that the mRNA expression level of UBR5 was significantly elevated in tumors compared with matched adjacent non-tumor tissue (Fig. 1a). Consistently, higher expression of UBR5 protein was also found in CRC tissues as indicated by Western blotting (Fig. 1b). We then examined UBR5 expression in 80 CRC tissues by immunohistochemistry (IHC) staining. The IHC results

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Fig. 1 Relative UBR5 expression in CRC tissues and its clinical significance. a The mRNA level of UBR5 is increased in CRC tissues compared with matched adjacent non-tumor tissues. *P \ 0.05. b Representative (left) and quantification (right) of the protein level of UBR5 in CRC tissues and corresponding non-tumor tissues. b-actin

was a loading control. *P \ 0.05. c Representative images (left) and quantification (right) of UBR5 staining in 80 paired CRC tissues and corresponding non-tumor tissues. Scale bar 50 lm. *P \ 0.05. d Kaplan–Meier survival rates for CRC patients with high (n = 53) and low (n = 27) UBR5 expression

revealed that UBR5 was overexpressed in 71.0% (57/80) CRC specimens detected, while weakly positive staining was observed in the adjacent non-tumor tissues (Fig. 1c). These results obviously suggested that UBR5 expression is upregulated in CRC tissues. Next, we analyzed the association between UBR5 expression and clinicopathological parameters in 80 CRC patients (Table 1). The results show that overexpression of UBR5 did not correlate with age, gender, tumor site, and size (P [ 0.05), but was significantly associated with advanced clinical stage (P = 0.002) and distant metastasis (P = 0.015). Further, we analyzed the association between UBR5 expression and the overall survival in these 80 CRC patients by Kaplan–Meier method. As shown in Fig. 1d, the results revealed that high level of UBR5 expression had a shorter survival time than patients who had low level of UBR5 expression (n = 80, P = 0.015), implicating that UBR5 may serve as a prognostic factor in CRC. Final multivariate analyses by using the Cox regression model revealed that UBR5 expression as well as TNM stage was an independent prognostic factor for overall survival (hazard ratio 1. 978, 95% CI 1.046–3.257, P \ 0.001) (Table 2).

Table 1 Clinicopathological correlation of UBR5 expression in 80 CRC patients

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Variables

All cases

UBR5 expression High

Low

P value

Age (years) B60

30

20

10

[60

50

37

13

0.483

Gender Male

46

31

15

Female

34

26

8

M0

60

47

13

M1

20

10

10

0.375

Distant metastasis 0.015*

Tumor size(cm) B5

30

18

12

[5

50

39

11

I–II

24

16

8

III–IV

56

41

15

0.085

TNM stage 0.002*

Location Colon

48

31

17

Rectum

32

26

6

* P value \ 0.05, statistically significant

0.107

Dig Dis Sci Table 2 Multivariate analysis model for overall survival to assess UBR5 expression in CRC and clinical features

Variables in the model

Overall survival; multivariate HR (95% CI)

P value

TNM stage (III–IV vs. I–II)

2.056 (1.256–3.879)

0.001*

UBR5 expression (high vs. low)

1.978 (1.046–3.257)

\0.001*

HR hazard ratio, 95% CI 95% confidence interval * P \ 0.05, statistically significant

UBR5 Promotes Growth of CRC Cells In Vitro and In Vivo To further explore the biological role of UBR5 in CRC, we initially generated stable clones with UBR5 overexpression from the less metastatic cell line SW480, which exhibits relatively low expression of UBR5 among CRC

Fig. 2 UBR5 promotes growth of CRC cells in vitro and in vivo. a Representative Western blot showing the protein level in different CRC cell lines. b The efficiency of UBR5 silencing and overexpression in CRC cell lines was measured by Western blot. b-actin was a loading control. c, d Representative results for cell proliferation rate were evaluated in UBR5-overexpressing (c) and UBR5 knockdown (d) CRC cells. *P \ 0.05; **P \ 0.01. e, f Growth curve of tumor

cell lines (Fig. 2a, b). Meanwhile, we knocked down UBR5 by stably expressing UBR5 shRNA in LOVO cells, which normally shows relatively high expression of UBR5 (Fig. 2a, b). As shown in Fig. 2c, the results indicated that UBR5-overexpressing cells showed a significantly higher in vitro proliferation rate than control cells. In contrast, silencing UBR5 obviously suppressed

volumes after the subcutaneous injection of UBR5-overexpressing (e) and UBR5 knockdown (f) CRC cells. *P \ 0.05. g, h At the experimental endpoint, tumors were dissected, photographed, and weighed. n = 6, *P \ 0.05 versus control. i, j Representative images (left) and quantification (right) of the C-caspase 3 and Ki-67 staining of the transplanted tumors. Scale bar 50 lm. *P \ 0.05

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Fig. 3 UBR5 induces cell cycle progression and suppresses cell apoptosis. a–d The percentage of cells in different phases of the cell cycle was determined by FACS analysis of UBR5-overexpressed (a, b) and UBR5 knockdown cells (c, d). *P \ 0.05. ns not significant. e– g Representative results (left) and quantification (right) of the FACS

analysis showing the effect of UBR5 knockdown (e, f) and UBR5 overexpression (g) on the apoptosis of CRC cells after treatment with or without 5-FU (100 lM). The percentage of the PI-negative/annexin V-positive cell population was determined. *P \ 0.05

growth of CRC cells in vitro, when compared to the control group (Fig. 2d). Then, we stably overexpressed or knocked down UBR5 in CRC cells subsequently implanted as a xenograft into nude mice. After 5 weeks of growth, we found that SW480/ Lv-UBR5 cells had significantly greater tumor growth, whereas LOVO/sh-UBR5 cells exhibited reduced tumor growth in nude mice, when compared to respective controls (Fig. 2e, f). Consistently, the average tumor weight in mice bearing SW480/Lv-UBR5 cells was increased by 42%, whereas mice bearing LOVO/sh-UBR5 cells were decreased by 57%, when compared to respective controls (Fig. 2g, h). Finally, we analyzed the expression of the apoptosis marker cleaved caspase 3 (C-caspase 3) and the proliferation marker Ki-67 in xenograft tumor tissues. As expected, LOVO/sh-UBR5 cells exhibited enhanced apoptosis (Fig. 2i) and much lower expression of Ki-67 (Fig. 2j) than did control cells, consistent with the in vitro results. Taken together, these findings suggest that UBR5 contributed to the proliferation and apoptosis of CRC cells in vitro and in vivo.

demonstrate that UBR5 overexpression was association with an increase in S-phase cells and a corresponding decline of the G1-phase cells (Fig. 3a, b), when compared to controls. In contrast, the results indicate that silencing UBR5 leads to an increased percentage of cells in the G1 phase, alone with a decrease in S-phase cells (Fig. 3c, d). In addition, we also analyzed the effects of UBR5 on CRC cell apoptosis. After knockdown UBR5 in LOVO cells, the FACS data showed an increased apoptosis rate compared with the control cells (Fig. 3e). As shown in Fig. 3f, knockdown of UBR5 significantly increased the number of apoptotic cells induced by 5-FU (an anticancer drug). Conversely, the FACS data indicate that the increased apoptosis rate induced by 5-FU in SW480 cells was partly abolished by UBR5 overexpression (Fig. 3g). Collectively, these data indicate that UBR5 promotes cell proliferation by facilitating cell cycle progression and suppressing cell apoptosis.

UBR5 Promotes Cell Cycle Progression and Suppresses Cell Apoptosis

UBR5 has been shown to directly interact with numerous proteins implicated in a wide variety of cellular processes [5]. Here, we found that UBR5 regulated the protein stability of ECRG4, which acts as a novel suppressor gene in many cancers [12, 15]. As shown in Fig. 4a, the protein level of ECRG4 was significantly decreased in UBR5-

To explore the mechanism by which UBR5 contributes to tumor cell growth, we analyzed changes of the cell cycle in UBR5-overexpressing or knockdown cells. The FACS data

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ECRG4 Contributes to the Effects of UBR5 in CRC Cells

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Fig. 4 ECRG4 contributes to the effects of UBR5 in CRC. a, b Western blot analysis for expression of ECRG4, ATMIN, and CDK9 in UBR5-overexpressing SW480 cells and UBR5-silenced LOVO cells. c Graphic presentation shows mRNA expression level of ECRG4 in UBR5-overexpressing and UBR5-silenced. ns not significant. d Western blot analysis for expression of ECRG4 in UBR5overexpressing and UBR5-silenced CRC cells treated with/without cycloheximide (CHX, 100 mg/ml) for 6 h. b-actin was used as a

loading control. e Representative Western blots show that UBR5 directly interacts with ECRG4 in LOVO cells. e Representative Western blots show that the ubiquitination of ECRG4 in UBR5 overexpression cells after treatment with or without MG132 (10 mM). f Western blot analysis for expression of ECRG4. b-actin was used as a loading control. G, The proliferation rate of LOVO cells were stably transfected with sh-UBR5 in the presence of sh-ECRG4. *P \ 0.05 versus sh-UBR5 group

overexpressing cells, whereas it was increased in UBR5 knockdown cells. Besides, our results also demonstrated that the protein level of ATMIN (a target of UBR5) was significantly decreased in UBR5-overexpressing cells, whereas it was increased in UBR5 knockdown cells (Fig. 4b). However, the protein level of CDK9 (a known target of UBR5) showed no significant difference in CRC cells after modulation of UBR5 expression compared with their respective control groups (Fig. 4b). The doubt could be explained by the fact that the ubiquitination pattern of CDK9 may be modified by other E3 ligase in CRC cells. Interestingly, the mRNA level of ECRG4 was not affected by suppression or upregulation of UBR5 (Fig. 4c). Thus, we speculated that UBR5 might regulate ECRG4 expression by post-translational modification of the ECRG4 protein. Then, we treated CRC cells with cycloheximide (CHX) inhibiting protein synthesis. After treatment with CHX, overexpression of UBR5 significantly reduced the protein level of ECRG4, whereas silencing UBR5 led to a pronounced increase in the level of ECRG4 protein (Fig. 4d). Furthermore, the Co-IP results showed that the endogenous

UBR5 and ECRG4 form a protein complex in LOVO cells (Fig. 4e). Moreover, our data indicate that overexpression of UBR5 dramatically increased the levels of ECRG4 ubiquitination (Fig. 4f). Finally, a rescue experiment was also performed by co-transfecting UBR5 shRNA and the lentivirus vector containing sh-ECRG4 into CRC cells (Fig. 4g). Indeed, we found that ECRG4 silencing enhanced the proliferative capacity of CRC cells, when compared with controls (Fig. 4h). The data show that the inhibitory effect of UBR5 knockdown on cell proliferation could be partially reversed by the introduction of shECRG4 in LOVO cells (Fig. 4h). Therefore, these results confirm that UBR5 contributes to CRC progression partly through destabilizing the tumor suppressor ECRG4.

Discussion Recent studies have demonstrated that UBR5 is upregulated and functions as a potential tumor promoter in several malignant tumors [16]. However, no information is

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currently available on the role of UBR5 in colorectal cancer. Here, we found that high UBR5 expression was closely associated with advanced clinical stage, distant metastasis, and shorter overall survival in CRC patients. Further multivariate analysis indicated that UBR5 and TNM stage were independent prognostic factors for overall survival in patients with CRC. Moreover, our findings proved that UBR5 contributed to the growth of CRC in vitro and in vivo. Thus, these data indicated that high UBR5 expression may represent a novel indicator of poor prognosis in CRC and may function as an oncogene in CRC progression. The E3 ligase UBR5 is a key regulator of the ubiquitin– proteasome system in cancer and development [17]. Recently studies have shown that high UBR5 expression promotes cell proliferation in several cancers [7, 9]. Consistent with these studies, our results also demonstrated that overexpression of UBR5 significantly promoted in vitro cell proliferation and in vivo tumor growth, whereas silencing UBR5 inhibited the growth of CRC cells. Our data present evidence that UBR5 promotes CRC cell proliferation by facilitating cell cycle progression and suppressing cell apoptosis. A previous study came to similar conclusions by showing that UBR5 down-regulates proapoptotic MOAP-1 and inhibits cisplatin-induced apoptosis in ovarian cancer [18]. Therefore, these findings, together with the high expression in CRC, indicate the tumorigenic function of UBR5 in CRC progression. UBR5 has been reported to target several other proteins for ubiquitination, including ATMIN [19], CDK9 [20], PEPCK1 [21], and GKN1 [7]. Especially, UBR5 negatively regulates TIP60’s stability through the proteasome pathway in cervical cancer [9]. In this study, we show that UBR5 directly binds to ECRG4 in CRC cells and this interaction contributes to the progression of CRC. In line with previous research, our findings confirmed that UBR5 regulates ECRG4 expression by increasing its ubiquitination and subsequent degradation. Our results also show that ECGR4 knockdown reverses the inhibiting effect of silencing of UBR5 on CRC cell growth. However, the precise molecular mechanism by which ECRG4 suppresses the growth of CRC cells remains to be further explored. Thus, these results demonstrated that the effects of UBR5 in CRC cells are at least partly through downregulation of ECRG4. In conclusion, these results show that UBR5 serves as an oncogenic protein in CRC and suggests that UBR5 can contribute to the progression of CRC by destabilizing ECRG4. Thus, UBR5 may be a potential therapeutic target for CRC treatment. Compliance with ethical standards Conflict of interest None.

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