The Role Of USP33 In Suppressing Epithelial-Mesenchymal Transition,Migration And Invasion Of Colorectal Cancer Cells

Colorectal cancer(CRC)is the carcinoma originated from colon or rectum,and is one of the most common tumor types.The morbidity of CRC in China is increasing in the past decades.The prognosis of CRC depends largely on its clinical stage,the overall survival of CRC patients with distant metastasis is less than 15%.The liver metastasis CRC(CRCLM)is the major death cause,and more than 20%patients has synchronous liver metastasis at the time of initial diagnosis.Even for the CRC patients without distant metastasis,about 30%patients will occur metachronous liver metastasis after surgical resection.The therapy for CRCLM patients is multi-disciplinary and depends on personal clinical features,and surgical resection of CRC as well as CRCLM is the best treatment strategy if possible.However,the intrahepatic recurrence is still high after primary surgery.Therefore,the treatment for CRCLM patients is the key aspect to improve the overall prognosis of CRC.The liver is the most common site of metastasis due to the drain via the portal circulation,and CRCLM is a complicated biological process.Cell adhesion,cell proliferation,migration,invasion and tumor cell microenvironment can all affect the metastatic progression.Even the detailed underlying molecular mechanisms of CRCLM need further elucidate,the role of chemokine and chemokine receptors are common recognized.Another important mechanism of tumor metastasis is the epithelial-mesenchymal transition(EMT).C-X-C chemokine receptor type 4(CXCR4)is one of the most important members of C-X-C chemokine receptor family,belonging to the G protein coupled receptors(GPCRs),which can be activated by several chemokines especially C-X-C motif chemokine 12(CXCL12,also named as SDF-1).Once the membrane receptor CXCR4 is activated,it can trigger the downstream extracellular signal-regulated kinase(ERK)signaling,thus regulated ERK-dependent EMT protein expression and the biological features of tumor cells.Similar with most GPCRs,the activated CXCR4 will undergo desensitization and internalization processes.The desensitization means the ligand-activated receptors lose signaling activity due to its phosphorylation,which can serve as a negative feedback.The internalization refers to the endocytosis of the activated-receptors,which is ?-arrestin dependent.The internalized receptors have two destinations,one is degraded in proteasome and the other is recycling to cell membrane.For the internalized receptors that can recycle to cell membrane,it will be dephosphorylated in the endocytosed endosome and resensitized,therefore ensuring the functional numbers of receptors on cell surface.Therefore,the balance of receptor desensitization,internalization and recycling is critical in maintaining GPCR functions.In addition,the receptor desensitization and internalization are regulated by multi-mechanisms.For example,the post-translational modifications(PTMs)of receptors and ?-arrestins can both modulate the process of receptor internalization,with phosphorylation and ubiquitination as two of the most important PTMs.Ubiquitin-specific protease 33(USP33)belongs to the family of ubiquitin-specific proteases(USPs),which can catalyze the deubiquitination process of ubiquitinated-proteins.USP33 was reported to participate in the internalization regulation of ?2-adrenergic receptor,another important GPCR family members.However,the expression and functional roles of USP33 in tumor progression is little known.Our study initially explored the expression patterns of USP33 in CRCLM patients,which revealed that USP33 was down-regulated in both CRC and CRCLM tumor tissues,compared with adjacent non-tumorous tissues.We analyzed the relationship between USP33 expression with patients'clinicopathological characters and prognosis.In addition,our results demonstrated that USP33 can inhibit the ?-arrestin2-mediated internalization of CXCR4,subsequently modulate the EMT protein expression and invasion capacity of colorectal cancer cells.In conclusion,our study not only verified the role of USP33 in regulating colorectal cancer metastasis,but also elucidate the involvement of ?-arrestin-dependent ERK signaling in cancer progression.Part I.The expression of USP33 in CRCLM patients and its association with clinicopathological characteristics[Objective]To investigate the expression patterns of USP33 in primary CRC tissues,metastatic CRCLM tissues,and adjacent non-tumorous tissues.To analyze the correlations between USP33 expression with patients' clinicopathological characteristics,as well as its role in predicting prognosis.[Materials and Methods]1.We collected 139 CRC patients with synchronous liver metastases or early metachronous liver metastases(defined as liver metastases occurring within 12 months of the primary CRC diagnosis),all of whom underwent surgical resection of primary CRC and CRCLM from January 2000 to January 2013 in Qilu Hospital,Qianfoshan Hospital,Shanxian Central Hospital or Yuncheng People's Hospital.Clinical information retrieved include gender,age,primary tumor location,primary tumor differentiation,primary tumor stage,preoperative CEA level,lymph node metastasis,extrahepatic metastasis;as well as the distribution,number,largest diameter,differentiation and resection margin of liver metastases.Paraffin-embedded specimens of tumor tissues and adjacent non-tumorous tissues were obtained from the Department of Pathology.Representative 1.5-mm tissue cores from each specimen were used to conduct a tissue microarray(TMA)analysis,and TMA samples were then cut into 4-?m sections slides for the later immunohistochemical(IHC)st,aining of USP33.All of the statistical data was calculated by SPSS 20.0 software(IBM).Continuous variables were analyzed with Spearman's rank correlation and Student's t-test,while categorical variables were analyzed using the ?2 test.2.Another 14 paired fresh-frozen tissues from patients with CRCLM were collected from 2014 to 2016,and stored in liquid-nitrogen until use.Total RNA from fresh-frozen tissue specimens or cell lines was extracted using TRIzol reagent,followed by RT-qPCR of USP33.3.A Western Blot assay was performed to measure the protein levels of USP33 in the 14 paired fresh-frozen tissues as described above.4.Retrospectively collected the patients' survival information.The overall survival(OS)was defined as the period from liver metastases resection to the date of death or the last follow-up;and the disease-free survival(DFS)was measured from the date of liver metastases resection to the date of recurrence or the date of death or the last day of follow-up.The Kaplan-Meier method and log-rank test were used to evaluate the prognostic value of USP33 expression and clinicopathological patient characteristics.Multivariate Cox proportional hazard regression models was used to identify the independent prognostic factors associated with OS or DFS.Only the variables significantly associated with OS or DFS by univariate analysis were used as covariates for the multivariate analysis.P<0.05 was considered as statistical significant.[Results]1.USP33 mainly located in the cytoplasm and was significantly down-regulated in both the CRC and CRCLM tissues,compared with adjacent non-tumorous tissues.USP33 low-expression in CRC and CRCLM were both correlated with advanced tumor stage and positive LN metastasis.Importantly,the USP33 expression in primary CRC,but not CRCLM,is associated with the number of liver metastases.2.The mRNA level of USP33 in tumor tissues was lower than that in adjacent non-tumorous tissues(9/14 in CRC tissues,and 12/14 in CRCLM tissues).3.The protein level of USP33 in tumor tissues was lower than that in adjacent non-tumorous tissues(10/14 in CRC tissues,and 12/14 in CRCLM tissues),which is consistent with the RT-qPCR results.4.Using Kaplan-Meier survival analysis,we identified that bilobar(P=0.001),multi-number(P=0.010),larger diameter(P=0.009),poor differentiation(P=0.018),and positive resection margin(P<0.001)of CRCLM were all correlated with poor OS.Moreover,the low-expression of USP33 in either CRC or CRCLM was significantly correlated with poor clinical outcome.Other prognostic factors for OS included LN metastasis and extrahepatic metastasis.Probable risk factors of recurrence were also evaluated by univariate analysis.The distribution(P=0.045),number(P<0.001),largest diameter(P=0.020),differentiation(P=0.047),and resection margin(P<0.001)of CRCLM were all predictive aspects for DFS.Besides,the primary CRC tumor stage(P=0.019),LN metastasis(P<0.001),and USP33 expression in CRCLM(P<0.001)were also associated with the disease relapse.Moreover,multivariate analysis verified that the low-expression of USP33 in CRCLM,but not in CRC,can serve as an independent prognostic factor for both OS and DFS.[Conclusions]1.USP33 is down-regulated in colorectal cancer and may function as a tumor suppressor.2.USP33 low expression is correlated with advanced tumor stage and invasive capacity.3.The expression level of USP33 in CRCLM can serve as an independent prognostic factor for both OS and DFS,Part ?.USP33 inhibits cell proliferation,migration and invasion of colorectal cancer cells by down-regulating the EMT process[Objective]As described above,the expression of USP33 was down-regulated in CRC,thus we aimed to further verify the role and function of USP33 in regulating the proliferation,migration and invasion of colorectal cancer cells.[Materials and Methods]1.The expression of USP33 in human CRC cell lines(LoVo,SW480,SW620)and NCM460 normal colonic epithelial cell line was tested by Western blot assay.2.HA-USP33 plasmids was constructed and USP33-siRNA was purchased.The LoVo cells were transfected by either HA-USP33,vector,USP33-siRNA or negative control siRNA(NC-siRNA).3.Stimulated the transfected LoVo cells with SDF-1,and monitored the effects of USP33 in regulating the morphological changes.The expression levels of EMT proteins were detected by Western blot.4.The transfected LoVo cells were stimulated with SDF-1,followed by MTT assay,wound healing assay and Transwell assay to test the effects of USP33 in modulating the biological characteristics of CRC cells.[Results]1.Western Blot results showed that NCM460 has the highest USP33 expression,which is consistent with the IHC results;LoVo cells showed moderate USP33 expression among all the cell lines.2.The plasmid of HA-USP33 was successfully constructed and verified by DNA sequencing.The transfection efficiency was examined by Western blot.3.The HA-USP33 transfection can significantly inhibit the EMT of LoVo cells by comparing the morphology of USP33-siRNA cells using light microscope.We found that USP33 overexpression can significantly down-regulate the protein level of ?-catenin,snail,slug,twistl,and N-cadherin,while increasing the E-cadherin expression.On the other hand,silencing USP33 exhibited reciprocal regulation effects towards the EMT proteins.4.Overexpression of USP33 resulted in a significant decrease of the proliferative and invasive capacity of CRC cells,as determined by MTT assay,wound healing assay,and transwell invasion assay.In contrast,USP33 knock-down showed positive regulation towards the cell viability and invasion.[Conclusions]1.USP33 can inhibit the SDF-1-induced EMT process of CRC cells.2.USP33 can inhibit the SDF-1-induced migration and invasion of CRC cells.Part ?.The molecular mechanisms of USP33 in suppressing colorectal cancer progression[Objective]Since the results above showed that USP33 plays important roles in regulating the EMT and invasion of CRC cells,we carried out more molecular experiments to further explore the underlying mechanism of its functions.[Materials and Methods]1.The expression of CXCR4 in tumor tissues and adjacent tissues was detected by IHC.2.The mRNA and protein levels of CXCR4 in different CRC cell lines were detected by RT-qPCR and Western blot,respectively.3.The inactive USP33 mutant,HA-USP33Cys:His(C214S/H683Q),was introduced by site-directed mutagenesis,and transfected into LoVo cells.4.LoVo cells transfected with HA-USP33Cys:His was tested with MTT and Transwell assays to see whether the enzymatic activity of USP33 is necessary in mediating CRC progression.5.The role of USP33 in regulating CXCR4 internalization and recycling was detected by cell membrane ELISA method.6.The immunoprecipitation experiment was performed to identify the complex formation between USP33 and ?-arrestin2.The effects of USP33 on ?-arrestin2 protein degradation was also tested by Western blot.7.The protein expression of ?-arrestin2 in tumor tissues and adjacent tissues was detected by IHC.8.The effects of USP33 on SDF-1-induced ERK signaling was tested by Western blot,and dynasore(an internalization inhibitor)was used to see whether USP33 function through mediating the internalization of CXCR4.9.Pretreated the USP33-siRNA transfected LoVo cells with dynasore,and stimulated with SDF-1,then carried out MTT and Transwell assays to verify whether the CXCR4 internalization can affect the tumorgenicity of CRC cells.[Results]1.We found that the expression of CXCR4 in tumor tissues was higher than that in adjacent non-tumorous tissues.2.The LoVo cells showed detectable endogenous CXCR4.The mRNA levels of CXCR4 in CRC cell lines were ranked as SW480>LoVo>SW620,and the protein levels were consistent with mRNA levels.3.The construction of HA-USP33Cys:His plasmid was verified by DNA sequence,and its transfection efficiency was detected by Western blot.4.In cells transfected with USP33oys:His,the internalization extent was similar with blank control group,meaning the USP33's enzymatic activity is fundamental in preventing receptor internalization.5.USP33 knock-down can increase the degradation of CXCR4,while USP33 overexpression can inhibit its degradation.Furthermore,we performed on surface ELISA to evaluate receptor internalization as well as the recycle ratio of internalized receptors,which interestingly demonstrated that USP33 knock-down can not only promote internalization,but also impair the recycling of CXCR4.6.The immunoprecipitation assay showed that USP33 can interact with ?-arrestin2 in an agonist-dependent manner.Moreover,USP33 knock-down can significantly increased the ubiquitination level of ?-arrestin2 under SDF-1 stimulation.However,the protein level of ?-arrestin2 was not affected by USP33.7.Although ?-arrestin2 was up-regulated in both primary CRC tissues and liver metastases,there was no significant correlation between the USP33 and ?-arrestin2 expression levels.8.The ERK signaling was prolonged and "right shift" in the cells transfected with USP33-siRNA,and dynasore pretreatment can impair this effect.9.Pretreatment with dynasore can attenuate the effects of USP33-siRNA in promoting cell proliferation and invasion.[Conclusions]1.The deubiquitination enzymatic activity of USP33 is critical for its function as a tumor suppressor in colorectal cancer.2.USP33 can down-regulate the ubiquitination level of ?-arrestin2,therefore decrease the ?-arrestin2's binding affinity towards CXCR4,but has no effect on its proteasome-degradation process.3.USP33 knock-down can significantly prolong SDF-1 induced ERK signaling in colorectal cancer cells by increasing the ubiquitination level of ?-arrestin2 and internalization of CXCR4,therefore enhance the carcinogenicity of cancer cells.