UCHL1 Promotes Chemoresistance to 5-Fluoruracil Based Adjuvant Chemotherapy and is a Prognostic Marker for Stage II Colon Cancer Patients

Background: 5-Fluoruracil based adjuvant chemotherapy after radical resection is recommended for stage II colon cancer patients with high risk of recurrence. Up to now, novel biomarkers still needed for better stratication for improving prognosis. Methods: Here we report that UCHL1 is an independent prognostic factor for stage II colon cancer patients and promotes chemoresistance both in vitro and in vivo. Results: Our study indicated that UCHL1 is signicant up regulated in 96 pairs of stage II colon cancer patients who received postoperative 5-FU based chemotherapy. Stage II colon cancer patients with high UCHL1 expression showed high recurrence rate after chemotherapy. Multivariate Cox regression analysis showed that UCHL1 is an independent prognostic factor for overall survival (P=0.008) and disease-free survival (P=0.001). 5-FU based chemoresistance is examined in colon cancer cell lines (RKO and LoVo) with down regulation of UCHL1 by cytotoxicity test. Down regulation of UCHL1 exhibited decreased cell viability, elevated cell apoptosis rate, increased G2/M-phase and elevated level of cleaved caspase 3 and PARP when treated with 5-FU. Furthermore, the results in xenograft model are consistent with results in vitro. Conclusions: UCHL1 potentially contributing to identify recurrence risk and predict the benet for postoperative 5-FU based adjuvant chemotherapy in stage II colon cancer patients.

Mutations (both hereditary and somatic) that contribute to adenoma formation or progression have been identi ed in a number of genes. Most signi cantly, mutations in genes involved in Wnt/β-catenin signaling, such as the adenomatous polyposis coli (APC) gene, cause colon cancer through the constitutive formation of a nuclear β-catenin/TCF transcription complex in colonic epithelial cells [8][9][10] .
Ubiquitin C-terminal hydrolase L1(UCHL1), also known as PARK5, belongs to the peptidase C12 family [11,12] . UCHL1 acts as either an oncogene or a tumor-suppressor gene depending on the cancer types, partially because its nature of the deubiquitinating activity that inhibits proteasome mediated degradation is diverse, by the deubiquitination of proteins themselves that are affected in a variety of tumor tissues [13][14][15] . Previous report showed that UCHL1 promotes uterine serous cancer cell proliferation and cell cycle progression [16] . UCHL1 acts as a candidate oncoprotein that promotes TGFβ induced breast cancer metastasis and chemoresistance. Also, UCHL1 upregulation by verapamil enhanced the reversal effect of verapamil on chemoresistance of hepatocellular carcinoma and promoted cell apoptosis [17,18] . Here, we examine the UCHL1 expression patterns and clarify the pathological signi cance and patients survival in stage II colon cancer. Then we evaluate the value of UCHL1 for predict response to 5-Fu based chemotherapy in stage II colon cancer, and determine whether UCHL1 expression is a useful marker for guiding individualized adjuvant chemotherapy after radical resection in stage II colon cancer patients.

Materials And Methods 1 Patients and tissue samples
A total of 96 tissue samples from stage II colon cancer patients with pathologically con rmed diagnosis were obtained immediately after surgery and frozen at -80℃ in liquid nitrogen before being deposited in the Shenzhen Longhua District Central Hospital from January 2019 to August 2019. All patients received 5-FU based chemotherapy after surgery and the pathologic veri cation of diagnosis and staging were summarized according to the National Comprehensive Cancer Network (NCCN) Practice guidelines.
Overall survival (OS) and disease-free survival (DFS) were de ned as the interval from the initial surgery to death and clinically recurrence or metastasis, respectively. Written informed consent was obtained from each patient before surgery and all study protocols were approved by the Ethics Committee for Clinical Research of Shenzhen Longhua District Central Hospital.

Immunohistochemistry
A total of 114 stage II colon cancer specimens were collected, xed in 10% neutral buffer formalin, and embedded in para n blocks. Tissue sections (4µm) were processed for immunohistochemical staining according to the Dako Envision System (Dako Glostrup, Denmark). Brie y, the sections were depara nized, rehydrated in serially graded ethanol, and heated 5 min in citric buffer (pH 6.0) once for antigen retrieval. Sections were immunolabeled with primary antibody UCHL1(1:200 dilution, ab27053, United Kingdom) at 4℃ overnight. Washed sections were incubated with goat anti-rabbit Envision System Plus-HRP (Dako Glostrup, Denmark). PBS was used as a negative control. Immunostaining scores were nally recorded by two experienced pathologists in a blinded fashion according to the staining intensity and extent of staining which was previously described [19] .

Western blot analysis
Total protein of frozen colon cancer and its paired normal mucosa specimens were extracted and lysed in RIPA buffer with protease inhibitors. Each 50µg aliquot of total protein were separated via 8% SDS-PAGE and transferred to nitrocellulose membrane. The membranes were blocked in 10% skim milk and incubated overnight with anti-UCHL1 (1:500 dilution, ab27053, United Kingdom). For in vitro experiments, Cell lysates were separated via 8% SDS-PAGE and transferred to nitrocellulose membrane. The membranes were blocked in 10% skim milk and incubated overnight with anti-cleaved Caspase-3, anticleaved PARP. Subsequently, all membranes were incubated for 1h with horseradish peroxidaseconjugated secondary antibody, and the speci c bands were collected nally.

Cell culture and lentivirus infection
The high expression of UCHL1 in RKO and LoVo colon cancer cell lines was observed in our previous study, so we choose these two cell lines for further functional tests. RKO and LoVo were purchased from American Type Culture Collextion (ATCC® Manassas, VA, USA). All cells were maintained in Dulbecco's modi ed Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen Corp, USA) and cells were incubated at 37°C in a humidi ed incubator containing 5% CO2. 5-FU was purchased from Sigma were added to each well after sixty hours. The 450nm absorbance of each well was determined on a microplate reader (Bio-Rad, USA). Cell viability was calculated according to the following formula: Cell viability (%) = A450 (test group) / A450 (control group) ×100%. IC50 (half maximal inhibitory concentration) was calculated from the dose -response curves. Approximately 5 × 10 6 RKO cells transfected with siR-UCHL1 or negative control were injected subcutaneously into the opposite anks of each mouse. When the tumor volumes were approximately 0.2cm 3 , a dose of 20mg/kg/day 5-FU was applied for intraperitoneal injection twice a week, total 3 weeks. Tumor dimensions were measured once 5 days. All nude mice were sacri ced after 3 weeks.
Tumor volume was calculated using the following formula: width 2 ×length×0.5. All of the animal procedures were conducted in accordance with the Shenzhen Longhua District Central Hospital Animal Care guidelines.

Results
1 Aberrant overexpression of UCHL1 in stage II colon cancer patients A total of 96 stage II colon cancer tissues were used to analysis UCHL1 mRNA expression. Randomly selected 4 paired stage II colon cancer tissues were used to analysis UCHL1 protein expression. As shown in Fig. 1A, the relative level of UCHL1 was signi cant up regulated in 96 stage II colon cancer tissues than in the matched non-cancerous mucosa (P < 0.001). Subsequent western blotting con rmed that the level of UCHL1 protein were also higher in the colon cancer tissues than in the paired non-cancerous mucosa (Fig. 1B).

Correlation between UCHL1 expression and clinicopathological characteristics of colon cancer
To further explore the correlation between UCHL1 and progression of colon cancer, UCHL1 expression patterns were determined by immunohistochemistry study in 114 stage II patients who accepted 5-FU based chemotherapy after radical resection. Results showed that the immunoreactive patterns of UCHL1 was predominantly positively identi ed in the majority of colon cancer specimens ( Fig. 2A, Table 1). Of the 114 subjects, correlation between UCHL1 expression and clinicopathological characteristics were demonstrated in Table 1. We observed that the overexpression of UCHL1 was closely correlated with depth of tumor invasion (P = 0.010), histologic differentiation (P = 0.001) and recurrence (P < 0.001). No correlations founds between UCHL1 expression and age, gender or tumor location status. Collectively, all these results indicated that UCHL1 possible plays critical role in stage II colon cancer patients.
3 Overexpression of UCHL11 associated with a poor prognosis in stage II colon cancer To assess the clinical value of UCHL1 expression in stage II colon cancer patients survival, Kaplan-Meier curves with a log rank test for overall survival (OS) and disease-free survival (DFS) were undertaken. The results indicated that there was a signi cant difference between UCHL1-positive and UCHL1-negative groups who developed primary colon cancer recurrence after 5-FU based chemotherapy. The recurrence rate was correlated with UCHL1 expression status, positive UCHL1 expression were associated with increased risk of recurrence (P = 0.001). As shown in Fig. 2B, patients with UCHL1-positive (weak and strong) expression had a remarkably lower OS and DFS rate than patients with UCHL1-negative expression (P < 0.001). The estimated mean OS time was signi cantly different between patients with UCHL1-positive and UCHL1-negative tumors (37.29 ± 19.225 and 54.12 ± 10.982 months, respectively, P < 0.001). The estimated mean DFS time was 33.21 ± 17.432 and 50.014 ± 14.352 months for subjects with UCHL1-positive and UCHL1-negative tumors (P < 0.001). In addition, univariate and multivariate hazard ratios for survival were calculated by Cox proportional hazards model analyses. In multivariate analysis using the clinicopathological variables such as T stage and differentiation grade, the expression of UCHL1 was an independent prognostic marker to predict patient outcomes (OS: hazard ratio: 3.504, 95% con dence interval: 2.246-6.807, P = 0.008. DFS: hazard ratio: 2.304, 95% con dence interval: 1.967-6.383, P = 0.001, respectively ( Table 2 and Table 3).

Down regulation of UCHL1 inhibits cell viability and chemoresistance in vitro
Chemoresistance is one of the important causes of failed therapy and results in recurrence in colon cancer patients. To determine the potential role of UCHL1 in chemoresistance, a lentiviral vector for inhibition UCHL1 gene were constructed and transfected in RKO and LoVo colon cancer cell lines. All experimental groups exposed to different concentrations of 5-FU treatment in vitro. CCK8 test indicated that down regulation of UCHL1 signi cantly inhibited cell viability of RKO and LoVo cells (Fig. 3A). The IC50 were signi cant decreased in RKO and LoVo cells (Fig. 3B).

Down regulation of UCHL1 sensitizes colon cancer cells to apoptosis
To further determine the potential function of UCHL1 in chemoresistance, cell cycle and apoptosis analysis were applied. Down regulation of UCHL1 showed a signi cant increase in G2/M-phase cells, and a reciprocal decrease in G0/G1-phase cells, compared to vector cells (Fig. 4A). The results we observed are augmented by the addition of 5-FU. These ndings indicated that at the time of 5-FU treatment, cells are blocked in a more chemosensitive stage of cell cycle. In addition, down regulation of UCHL1 had little effects on cell viability in the absence of 5-FU. However, the ability to promote cell apoptosis were observed when cells treated with 5-FU (Fig. 4B, 4C). To further explore the molecular mechanisms underlying the anti-apoptotic role of UCHL1, we analyzed two biomarkers of apoptosis, poly adenosine diphosphate ribose polymerase (PARP) and cleaved caspase 3. After 48 hours treatment with 10 µM 5-FU, cleaved PARP and caspase 3 were detected in the siR-UCHL1 cell lines (Fig. 5). Compared with control cells, which were resistant to apoptosis, the majority of PARP and caspase 3 proteins were uncleaved and remained full length. All in vitro ndings showed that UCHL1 plays important roles in protect colon cancer cells from 5-FU induced apoptosis.
6 Down regulation of UCHL1 can inhibit colon cancer growth and enhance 5-FU based chemotherapy sensitivity in vivo.
To evaluate the effect of UCHL1 on tumorigenesis in vivo, RKO cells were subcutaneously implanted in BALB/c mice. Cells transfected with the siR-UCHL1 or Vector control were injected into the opposite anks of each animal which were treated with 5-FU or not. The mice were euthanized and the tumor growth curve and harvested tumor weights were shown in Fig. 6. Down regulation of UCHL1 results in signi cantly reduced average tumor size and weight compared to that of control tumors. An even more signi cant reduction was indicated in the siR-UCHL1 group treated with 5-FU. Taken together, these ndings con rmed that down regulation of UCHL1 can inhibit colon cancer progress and enhance 5-FU based chemotherapy sensitivity in vivo.

Discussion
Colon cancer is one of the major malignant diseases detrimental to health and chemotherapy resistanceis hot researched topic at present [20,21] . Our study support the hypothesis that UCHL1 could serve as a prognostic indicator of stage II colon cancer patients who treated with 5-uorouracil based adjuvant chemotherapy following curative surgery. Moreover, we investigated the impact of UCHL1 inhibition on chemoresistance in colon cancer cells. We clearly showed that UCHL1 inhibition confers a better phenotype and improved 5-FU based chemosensitivity in colon cancer cells.
UCHL1 is a widely studied deubiquitinase as it has been described in both neurodegenerative disorders and varying malignancies [22,23] . Depending on tumor types, some studies report UCHL1 as an oncogene, whereas others discuss its role as a tumor suppressor gene as a result of CpG island methylation [24] .
Previous report showed that UCHL1 acts as a candidate oncoprotein that promotes TGFβ induced breast cancer metastasis and chemoresistance. Inhibition of UCHL1 offer potential forms of treatment for invasive carcinomas including EBV-positive malignancies [25] . Based on the previous study, we further revealed that UCHL1-positive expression associated with poor prognosis in stage II colon cancer patients, and down regulation of UCHL1 could inhibit 5-FU based chemoresistance and sensitize colon cancer cells to apoptosis. These ndings showed that UCHL1 promote the biological activities of stage II colon cancer patients after surgery, even though treated with 5-FU based chemotherapy.
Up to now, the partial value of adjuvant chemotherapy for stage II colon cancer patients remains controversial. Stage II colon cancer patients were considered for chemotherapeutic therapy if, based on clinical and pathological evaluation, they are deemed to be at a high risk of relapse [26][27][28] . NCCN Guidelines indicated that patients of stage II with high risk factors, such as poor histological type, few examined lymph nodes, intestinal obstruction and vascular invasion, are likely to speci cally bene t from chemotherapy [29] . In our present research, stage II patients with UCHL1-positive expression showed increased risk of tumor recurrence when treated with 5-FU after radical operation. In addition, patients with UCHL1-positive expression had a remarkably lower OS and DFS rate, was an independent prognostic marker to predict patient outcomes. Our data showed that UCHL1 possess potential to predict 5-FU susceptibility in patients with stage II. These information could helpful for identify target therapeutics to patient subgroups with highest risk for disease recurrence. 5-FU based chemotherapy, was previously approved for rst or second line treatment of patients with colon cancer. Capecitabine is widely used in the therapy of stage II colon cancer patients, which is an oral drug of Fluorouracil (FU) [27] . 5-FU induced cancer cells apoptosis throuth DNA damage and growth inhibition. Chemotherapy resistance could result from multiple factors, including enhanced drug inactivation, mutation of drug target and defects of mismatch repair genes [30,31] . Our data showed that down regulation of UCHL1 signi cantly inhibited cell viability of RKO and LoVo cells, the IC50 of RKO and LoVo cells were signi cantly decreased by UCHL1 inhibition. Therefore, down regulation of UCHL1 could contribute to chemosensitivity of 5-FU based thermotherapy. Moreover, down regulation of UCHL1 resulted in cellular arrest in G2-M stage, a more chemosensitive stage of cell cycle. So, cells were arrested at a more chemosensitive stage of cell cycle. Molecular biology of tumors revealed that the imbalanced cell proliferation and cell apoptosis is an important mechanism of the tumorigenesis and progression [32][33][34] . In our study, UCHL1 inhibition signi cantly increased the extent of 5-FU induced apoptosis of colon cancer cells. To further explore the mechanism by which UCHL1 mediates chemoresistance, we examined two apoptosis markers, PARP and caspase 3 cleavage [35] . In UCHL1 down regulation cells treated with 5-FU, signi cantly more cleaved PARP and caspase 3 were detected compared with control cells. These ndings showed that UCHL1 could confer chemoresistance through anti-apoptotic pathways. Further in vivo study indicated that tumor growth and weights were signi cantly decreased in UCHL1 inhibition tumor xenografts, especially when treated with 5-FU. These results were consistent with previous in vitro results and suggest that UCHL1 is a critical protein responsible for 5-FU based chemoresistance.
In conclusion, UCHL1 can be used to effectively evaluate and distinguish recurrent risk in stage II colon cancer patients. UCHL1 abnormal expression decreases 5-FU susceptibility in colon cancer cells, which could contribute for guiding individualized chemotherapy bene t in stage II colon cancer patients after complete mesocolic excision, CME radical operation.

Conclusions
UCHL1 potentially contributing to identify recurrence risk and predict the bene t for postoperative 5-FU based adjuvant chemotherapy in stage II colon cancer patients.     Both the overall survival rate and disease-free survival rate of UCHL1-positive patients were signi cantly lower than that of the UCHL1-negative patients (log-rank test, P<0.001).