Proteomic Identification Of PRL-3 Associated Proteins: Role Of Stathmin In Colorectal Cancer

Colorectal cancer (CRC) is a common malignant tumor in the world. The incidence and death rate of CRC in China is increasing year by year during the past decade. Metastasis is the main cause affecting the therapeutic efficacy and leading to the death of patients with CRC. However,the mechanims of metastasis of CRC is still not well-known. To find genes associated with metastasis and elucidate their functions in CRC will be helpful for clinical diagnosis, prognosis analysis and possible target treatment of CRC.PRL-3 (Phosphatase of Regenerating Liver-3), a key gene associated with progression and metastasis of CRC, belongs to a protein tyrosine phosphatase family with only there members like PRL-1, PRL-2 and PRL-3. PRL-3 is a small molecule about 19KD. Many evidences have been reported in support of the role of PRL-3 in progression, metastasis and prognosis of CRC. PRL-3 is overexpressed in many kinds of cancer, such as ovarian cancers, gastric cancers, human gliomas, non-small cell lung cancer, and nasopharyngeal carcinoma. However, the role of PRL-3 in tumor progression and metastasis is still not very clear.PRL-3 was suggested to be involved in several kinds of signal pathways. PRL-3 regulates integrin/Src signaling pathway, Rho family GTPases, Ang-II signaling, Cell cycle et al. Enhanced Src signaling and PI-3K/Akt signaling contribute to epithelial msenchymal transition (EMT). Recently, we find that PRL-3 promotes EMT by regulating cadherin directly. PRL-3 is also an important cell cycle regulator and a direct p53 target gene. PRL-3 regulates invasion and metastasis by directly affecting the cytoskeleton and also through the transcriptional regulation of target genes. But, the proteins regulated by PRL-3 are still largely unknown.Several strategies can be used to find out the novel targets of PRL-3. The first one is to screen for PRL-3 interacting proteins. Integrin a 1 was first reported as a new interacting protein of PRL-3. Using this strategy, we identified CDH22 as a new PRL-3 interacting protein. Yeast two hybrid system and co-immunoprecipitation assay are two basic methods to find PRL-3 interacting proteins. The second strategy is based on proteomic methods to find out a great number of proteins regulated by PRL-3. Using this strategy, ezrin, cytokeratin 8 and the elongation factor 2 (EF2) are also found as substrates or interacting proteins of PRL-3.In order to further understand PRL-3 functions and its role in progression or metastasis of CRC, we performed proteomic analysis to detect changes in the protein levels in two kinds of cell models with PRL-3 transient or stable knockdown. We identified 39 differential spots regulated by PRL-3 using MALDI-TOF MS technique. We found that Stathmin, a key oncoprotein, was regulated by PRL-3Stathmin is a small molecule phosphoprotein about 17KD. It belongs to an unstable microtubule protein, and is associated with tumor invasion and metastasis. In this paper, we study the role of Stathmin in CRC, and we found a new link between PRL-3 and Stathmin, which contributed to progression and metastasis of CRC.Four groups were included in the following experiments:human colorectal cancer cell line SW480 as a control, SW480 cell line with PRL-3 transient knockdown for 24 hours or 48hours, SW480 cell line with PRL-3 stable knockdown. The cell pellets were lysed in sample buffer (7M urea,2M thiourea,0.2%(w/v) Bio-Lyte, pH 3-10,65mM DTT,4%(w/v) CHAPS) by sonication on ice. Lysates were centrifuged at 14 OOOxg for 1h. The supernatants used for 2-DE were stored at-80℃. Protein concentration was determined by Bradford method. Differentially expressed proteins were identified using 2D gel electrophoresis and mass spectrometry.2D gel electrophoresis was performed using IPG strip (17cm, pH 3-10, NL). Proteins were separated according to charge, and molecular weight. The gels were then underwent silver staining to visualize proteins. Images were obtained by scanning gels with scanner and analyzed using the PDQuest software v7.1.1. Differentially expressed spots were excised for in-gel digestion. Peptide mass mapping was performed by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF/TOF MS).Fresh or paraffin-embedded tissue samples from CRC patients at Nanfang Hospital in our university were used in the study. Clinicopathological classification and staging of these samples were performed according to the General Rules for Clinical and Pathological Studies on Cancer of the Colon, Rectum, and Anus along with the International Union Against Cancer classification. Immunohistochemical staining of protein expression of Stathmin in 149 human tissue samples of CRC was done using a Dako EnVision System. The sections were incubated overnight with the rabbit monoclonal anti-stathmin at a dilution of 1:500. The immunohistochemically stained tissue sections were analyzed separately by two pathologists without knowing the patients' clinical characteristics. Staining for Stathmin was assessed using a relatively simple, reproducible scoring method. The staining intensity was scored on a scale of 0 to 3 as negative (0), weak (1), medium (2), or strong (3). The extent of the staining, defined as the percentage of positive staining areas of tumor cells in relation to the whole tumor area, was scored on a scale of 0 to 4:0(0%),1(1-25%),2(26-50%), 3(51-75%) and 4 (76-100%). An overall protein expression score (overall score range, 0-12) was calculated by multiplying the intensity and positivity scores.Different human colorectal cancer cell lines or colorectal tumor tissues were homogenized in RIPA lysis buffer (50mM Tris containing 150mM NaCl,0.1% SDS, 1% Triton X-100,1% sodium deoxycholate, pH 7.2) with 0.2% protease and phosphatase inhibitor cocktail on ice for 15 min and centrifuged at 14,500×g for 30 min. The protein concentration of the supernatants was determined by BCA Assay, and aliquots of the protein samples were stored at-80℃. Equal amounts of proteins were separated electrophoretically on 13% SDS/polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (PVDF). The membrane was incubated at 4℃overnight with an anti-PRL-3 rabbit polyclonal antibody (1:100) or an anti-stathmin rabbit monoclonal antibody (1:20,0000).Expression of PRL-3 or Stathmin was determined with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (1:5000) and enhanced chemiluminescence. An anti-P-Actin goat monoclonal antibody (1:1000) was used to confirm equal loading.The human Stathmin-specific primers was cloned into the eukaryotic green fluorescent protein expression vector pEGFP-C1, the recombinant plasmid of pEGFP-C1-Stathmin was identified by PCR, restriction enzyme digestion analysis and DNA sequencing.The pEGFP-C1-Stathmin plasmid was transfected into SW480 cells and HT29 cells. Stathmin expression in transfectant was determined by Western Blot. Four different stathmin-specific short hairpin RNAs were cloned into pGPU6/GFP/Neo siRNA expression vector. After identification by PCR analysis and DNA sequencing, the recombinant vectors were transfected into colorectal cancer cells SW620 and LOVO,then Stathmin expression were identified by Western Blot.Effects of Stathmin overexpression and knock-down on cell proliferation was assessed by MTT assay, plate colony formation assay in vitro. Effects of Stathminoverexpression and knock-down on cell motility and migration were assessed in vitro by Transwell chamber and adhesion assay. SW480, Lovo, HT29 and SW620 cells were seeded on glass coverslips, fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in phosphate-buffered saline. Cells were stained using mouse polyclonal anti-PRL-3 or rabbit monoclonal anti-Stathmin, followed with the appropriate FITC-labeled donkey anti-rabbit IgG or TRITC-labeled donkey anti-mouse IgG/DyLight594 secondary antibodies.Approximately lml of whole cell lysate or tissue extract (about lmg) were pre-cleared using sepharose-coupled rabbit or mouse IgG at 4℃for 1h, then centrifuged at 12,000×g for 3min. Protein G Plus/Protein A Agarose and 5μg of primary antibody anti-PRL-3 or an anti-Stathmin rabbit monoclonal antibody were added to the supernatant, mixed and incubated at 4℃overnight. The immune complexes were pulled down with protein G Plus/Protein A Agarose. The sepharose beads were washed five times with PBS and then proteins were eluted by incubation in 2×SDS-loading buffer at 100℃for 5 min and aliquots were subjected to SDS-PAGE and western blot analysis. A rabbit monoclonal antibody against Stathmin or a rabbit polyclonal antibody against PRL-3 was used for western blot analysis. 4.3 Immunofluorescence detection of Expression of MTs in CRCsSW480, Lovo, HT29 and SW620 cells with PRL-3/Stathmin overexpressed or knocked-down were seeded on glass coverslips, fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in phosphate-buffered saline. Cells were incubated with mouse anti-a-tubulin antibody or anti-acetylated-α-tubulin at 4℃overnight after blocking with 1% BSA for 30min. Subsequently, the cells were incubated with TRITC-labeled donkey anti-mouse IgG/DyLight594 for 1h at 37℃. Processed coverslips were mounted in 75% glycerol/PBS mounting medium. Fluorescence was analyzed on confocal laser scanning microscopewith FV-10 ASW 1.7 Viewer image analysis software. We successfully identified 39 differential expression spots by two-dimensional gel electrophoresis and MALDI-TOF MS methods. Among these spots,21 proteins were down-regulated in both PRL-3 transient and stable knockdown clones,9 proteins were up-regulated in both PRL-3 transient and stable knockdown clones; we also observed 3 proteins were only down-regulated in PRL-3 stable knockdown clone and 6 proteins were only up-regulated in PRL-3 stable knockdown clone.Using functional distribution and category enrichment analysis of Gofact, we found that the proteins involved in process of cell cycle, cell death, biosynthesis, protein biosynthesis and translation were significantly enriched in these identified PRL-3 associated proteins. According to the components of these proteins, intracellular location including cilium, cytoplasm, cytosol, microtubule organizing center, and cytoskeleton were significantly enriched.we analyzed stathmin protein levels in an independent set of 149 paraffin-embedded, archival primary CRC tissues by immunohistochemical staining. Stathmin protein was localized in the cytoplasm of cancer cells. Higher expression of stathmin was observed in CRC tissue compared with that in normal counterparts. We found stronger expression of stathmin was found in both metastasis in lymph nodes and in livers. No significant association were found between stathmin expression and age, gender, tumor size, tumor site (P>0.05). Interestingly, we observed that stathmin expression was positively correlated with tumor differentiation(x2=6.656,P=0.010), tumor invasion(χ2=7.762,P=0.000),lymph node status(χ2=33.888,P=0.000), Dukes classification(χ2=37.480,P=0.000)and TNM staging (χ2=42.155,P=0.000) of CRC patients. Using Kaplan-Meier analysis method, we found that the protein expression of stathmin in CRC was significantly correlated with overall survival (χ2=53.205,P=0.000) of CRC patients. To determine whether the expression of stathmin was an independent prognostic factor of outcomes, multivariate survival analysis including invasion, differentiation, Dukes classification, TNM staging and stathmin expression, was done. Results showed that the expression of stathmin protein was a potential independent prognostic factor of outcomes of CRC patients(χ2=48.012,P=0.000).Western Blot showed that expression of stathmin was impaired when PRL-3 was transiently and stably knocked down; meanwhile,we performed western blot analysis to measure the protein expression of stathmin in fresh CRC tissue samples and their paired normal mucosal counterparts. We found that protein expression of stathmin was higher in most of the primary CRC tissue samples than that in their normal counterparts. Interestingly, protein expression of stathmin in primary tumor samples with metastasis (mCRC) is stronger that that in primary CRC tissues without metastasis (nmCRC). In CRC cell lines, strong expressions of stathmin were found in SW620, Lovo, HCT115, relatively low expressions of stathmin were observed in SW480, HT29 and LS174T. In our following functional study of stathmin, we study the effects of stathmin in CRC by downregulating expression of stathmin in SW620 or Lovo and by upregulating expression of stathmin in SW480 or HT29. 3 Stathmin Promotes Proliferation, Sdhesion And Migration of Human CRC CellsAccording to expression levels of stathmin in CRC cell lines, we study the effects of stathmin in CRC by down-regulating expression of stathmin in SW620 or Lovo and by up-regulating expression of stathmin in SW480 or HT29. A significantly increased proliferation detected by in vitro MTT assay was found in SW480 (F=22.236,P=0.000)and HT29(F=93.138,P=0.000) cells after stathmin expression in these cells was up-regulated. An impaired proliferation was found in SW620(F=16.791,P=0.000) and Lovo(F=19.599,P=0.000) cells after stathmin expression in these cells was down-regulated. Stathmin overexpression in SW480(F=113.958,P=0.000) and HT29 (F=33.477,P=0.000)cells had a significant enhanced ability to form colonies in plates; meanwhile, stathmin knockdown in SW620(F=30.258,P=0.000) and Lovo(F=31.172,P=0.000) cells had a decreased ability to form colonies in plates.Both gain-of-function and loss-of-function analyses revealed that stathmin promoted adhesion and migration of CRC cells(P=0.000).Stathmin MT-depolymerizing activity is negatively regulated by stathmin phosphorylation. As a phosphatase, PRL-3 may be involved in stathmin phosphorylation. Our co-immunoprecipitation assays in different CRC cell lines and CRC tissues revealed that PRL-3 could directly interact with stathmin. Co-localization of stathmin and PRL-3 in SW480, SW620, HT29 and Lovo cells was also found. Taken together, our results show that PRL-3 can interact directly with stathmin in the CRC cell systems.As we know, stathmin is a MT destabilizing protein. We observed the effects of PRL-3 or/and stathmin overexpression on distribution and expression ofα-tubulin. In control CRC cells, the distribution ofα-tubulin is dispersed into whole cells. However,α-tubulin aggregates locally after stathminWT or PRL-3 was up-regulated in two human CRC cell line SW480 and HT29. No significant change of distribution ofα-tubulin was observed after stathminWT or PRL-3 was down-regulated in two human CRC cell line Lovo and SW620.Stable MTs can be distinguished by a variety of posttranslational modifications, such as acetylation, poly-glutamylation, and detyrosination. Using specific acetylation antibody forα-tubulin, we explored the amount of stable MTs after stathminWT was up-regulated or down-regulated in human CRC cell lines. Expression of stathmin/EGFP significantly decreased the amount of cellular acetylated/stable MTs detected by acetylationα-tubulin. Consistently, increased the amount of cellular acetylated/stable MTs was observed after stathmin was knocked down in CRC cell lines Lovo and SW620. PRL-3 overexpression or knockdown led to similar effects as stathmin overexpression or knockdown induced. Westernblot analysis showed the similar result as Immunofluorescence detection. Based on the confirmed interaction between PRL-3 and stathmin, we concluded that interaction between PRL-3 and stathmin led to MT destabilization of CRC cells, which contributed to progression and metastasis of CRC.1. Thirty-nine PRL-3 associated proteins were screened and identified successfully. Proteins involved in process of cell cycle, cell death, biosynthesis, protein biosynthesis and translation were significantly enriched in these identified PRL-3 associated proteins.2. The expression of Stathmin/OP18 in CRC tissues is much higher than that in normal counterparts. Stathmin expression in metastatic CRC tissues is significantly higher than that in non-metastatic CRC tissues. It can be used as a potential independent predictor of the prognosis of CRC patients. Overexpression of Stathmin is related to tumor invasion, differentiation, Dukes classification, TNM staging of CRC patients. The expression of stathmin protein is a potential independent prognostic factor for outcomes of CRC patients.3. Stathmin/OP18 promotes proliferation, migration and adhesion of colorectal cancer cells.4. Interaction between PRL-3 and stathmin leads to MT destabilization of CRC cells, which contributes to progression and metastasis of CRC.