The Roles Of CacyBP/SIP Nuclear Translocation In Colon Cancer Cell

The CalCyclin Binding Protein (CacyBP) was firstly found in the cytosolic fraction of Ehrlich ascites tumor cells interacting with S100A6 (calCyclin) at a physiological range of Ca²⁺ concentration in 1998. Three years later, Siah-1 Interacting Protein (SIP) was confirmed as a human ortholog's CacyBP. Hence, CalCyclin Binding Protein was formally named as CacyBP/SIP.Further investigation showed that it could also bind other S100 proteins such as S100A1, S100A12, S100B and S100P. Recently, CacyBP/SIP was found to be involved in the development and differentiation of cells. Au et al. reported that overexpression of CacyBP/SIP promotes the differentiation and DNA synthesis in H9C2 cells, primary rat cardiomyocytes. The study of Yang et al. also revealed that progesterone (P4) and 17h-estradiol (E2) increase the expression of CacyBP/SIP gene. Hence, CacyBP/SIP was believed to participate in the regulation of apoptosis, and play an important role in mouse endometrial events such as pregnancy establishment. Interestingly, CacyBP/SIP could be translocated into nucleus and phosphorylated when Ca²⁺ concentration was changed in neurons and neuroblastoma NB-2a cells. This phenomenon has also been observed in retinoic acid-induced neuronal differentiation of neuroblastoma SH-SY5Y cells. However, the significance of CacyBP/SIP nuclear translocation is unknown. So we investigated whether CacyBP/SIP nuclear translocation might influence the function of colon cancer cells in the present work.【Objectives】(1) Establishment and Characterization of CacyBP/SIP Monoclonal Antibody. (2) To detect CacyBP/SIP protein expression in normal and malignant human tissues. (3) To investigate the founction of CacyBP/SIP in colon cancer by expressional and functional studies. (4) To examine the possible mechanisms of colon cancer induced by CacyBP/SIP nuclear translocation.【Methods】(1) The monoclonal antibodies against CacyBP/SIP were established with the lymphocyte hybridoma technology, and identified the corresponding MAbs of specificity and sensitivity with Western Blot and ELISA. (2) Purify the MAbs with caprylic acid-saturated ammonium sulfate and detecting the expression of CacyBP/SIP in normal and tumor tissue using immunohistochemistry. (3) The subcellular location and expression of CacyBP/SIP in normal colon tissues, colon cancer tissues, colon adjacent tissues and colon cancer cells were determined by immunohistochemistry assay and Western Blot. (4) Full-length vector, deletion mutants and siRNA vector of CacyBP/SIP were constructed. (5) The subcellular location of CacyBP/SIP in colon cancer cells induced by gastrin was observed by immuneofluorescence and Western Blot. (6) The effects of CacyBP/SIP nuclear translocation on the proliferation of colon cancer cell lines were respectively investigated by MTT assay, colony formation assays and cell cycle analysis. (7) The expression levels and the activity of cell cycle proteins after CacyBP/SIP nuclear translocation were determined by Western Blot, Cdk2 kinase assays and co-IP. (8) Detecting the changing of cell cycle protein with protease inhibitor MG132. (9) The location of truncation mutant of CacyBP/SIP induced by gastrin with confocal laser microscope. (10) The expression level of cell cycle protein of colon cancer cell transfecting truncation mutant of CacyBP/SIP with Western Blot and co-IP.【Results】1.Establishment and Characterization of CacyBP Monoclonal Antibody.Three hybridoma clones secreted MAb specific to the CacyBP/SIP protein were obtained. Immunoglobulin subclass determination showed that the MAbs were IgG1/κtype. Their ascites potency was 1×10^-7. Western blot and ELISA showed that the MAbs against CacyBP/SIP could recognize CacyBP/SIP protein in both native and denatured forms. These MAbs would act as a usefull tool for the detection of CacyBP/SIP protein in future studies.2. Expression of CacyBP/SIP protein in normal and malignant human tissues.CacyBP/SIP protein expression profiles in a broad range of human normal tissues and carcinomas were analyzed by immunohistochemistry staining with anti-CacyBP/SIP monoclonal antibody produced in our laboratory. CacyBP/SIP was generally localized in the cytoplasm/nucleus. Positive staining of CacyBP/SIP was found in brain, heart and lymph node. Weak stain was shown in rectum, kidney, prostate and esophagus. No CacyBP/SIP was detected in other normal tissues. However, CacyBP/SIP was ubiquitously detected in all kinds of tumor tissues, especially highly expressed in nasopharyngeal carcinoma, osteogenic sarcoma and pancreatic adenocarcinoma. Our results suggested that CacyBP/SIP may play important roles in tumorigenesis of human tumors.3. CacyBP/SIP expression in colon adencarcinoma tissues.To assess the biological roles of CacyBP/SIP in tumor progression, we firstly did immunohistochemistry on surgically removed colon tumors and their benign counterparts. CacyBP/SIP expressions were detected in the cytoplasm/nuclear of colon cancer tissues, with the positive rate 51% of colon adencarcinoma via 26% of colon adjacent tissues (p<0.05). In contrast, CacyBP/SIP stain was not detectable in 10 cases of normal colon tissues. The specificity of CacyBP/SIP immunoreactivity in tissues was validated by Western Blot analysis in the colon cancer and adjacent normal tissues taken from 4 patients. The result showed that CacyBP/SIP was overexpressed in colon cancerous tissues but not detectable in adjacent normal tissues. CacyBP/SIP protein was also present in the colon cancer cell lines HT29 and SW480. These results provide evidence that CacyBP/SIP expression level may be positively correlated with colon cancer onset or progression of the cancer.4. Nuclear translocation of CacyBP/SIP in colon cancer cells.It was well known that gastrin is a carcinogen in triggering colon cancer and it induces the intracellular Ca²⁺ mobilization. Others studies showed that CacyBP/SIP could be translocated into nucleus when Ca²⁺ concentration was changed. So we surmised that gastrin may induce CacyBP/SIP nucleus translocation by increasing the intracellular Ca²⁺ concentration. To elucidate the effect of gastrin on the intracellular distribution of CacyBP/SIP, we analyzed cultured colon cancer cells before and after gastrin stimulation. In unstimulated cells, CacyBP/SIP was distributed throughout the cytoplasm and CacyBP/SIP could translocate to the perinuclear region upon stimulation of gastrin. These phenomena were also observed with Western Blot.Two CacyBP/SIP-specific siRNA vectors, named CacyBP/SIPsi1 and CacyBP/SIPsi2 were designed and constructed. After cell transfection and G418 screening, CacyBP/SIPsi1 could down-regulate the expression of CacyBP/SIP in HT29 and SW480 effectively. Then cells stably transfected with CacyBP/SIPsi1, HT29-CacyBP/SIPsi1 and SW480-CacyBP/SIPsi1 were chosen for further cellular assay. In transfecting cells, after gastrin stimulation, CacyBP/SIP was distributed throughout the cytoplasm by immunofluorescent and wasn't detected in the cell nuclear by Western Blot. It was shown that gastrin couldn't induce CacyBP/SIP nuclear translocation after its expression was surpressed.5. CacyBP/SIP nuclear translocation promotes proliferation and cell cycle progression of colon cancer cells.To explore the effect of CacyBP/SIP nuclear translocation induced by gastrin on colon cancer cells proliferation, MTT assay and colony formation assay were used. The proliferation of HT29 and SW480 cells were enhanced by exogenous administration of gastrin(P< 0.05). Furthermore, CacyBP/SIP nuclear translocation after stimulation by gastrin dramatically enhanced anchorage-dependent growth as indicated by colony formation in flat (P<0.05). The cell cycle profile of colon cancer cells treated with gastrin was characterized as decreased percentage of cells in the G1 phase of the cell cycle.In HT29-CacyBP/SIPsi1 and SW480-CacyBP/SIPsi1 transfecting cells, MTT assay showed that proliferation was not significantly changed by exogenous administration of gastrin. At the same time, no enhanced anchorage-dependent growth was observed in transfected cells as indicated by colony formation in flat after stimulation by gastrin. Cell cycle analyses showed that no change appeared in the percentage of cells in the G1 phase of the cell cycle in transfected cells treated with gastrin. So, gastrin could enhance colon cancer cells proliferation via CacyBP/SIP nuclear translocation.6. CacyBP/SIP increasing expression of Cyclin E and decreasing the leval of P27^kip1.To correlate the effect of CacyBP/SIP on cell cycle progression with some molecular effectors of the restriction point, HT29 and SW480 cells were first synchronized with nocodazole. After stimulation by gastrin, the result showed that CacyBP/SIP induced a marked decrease of P27^kip1 expression level and increase of Cyclin E expression level. In HT29-CacyBP/SIPsi1 and SW480-CacyBP/SIPsi1 cells, no significant changes in protein levels of P27^kip1 and Cyclin E were observed. In comparison to cells without gastrin treatment, cells with gastrin treatment displayed less P27^kip1 bound to Cdk2 and elevated Cdk2 kinase activity. These results suggested that P27^kip1 participated in CacyBP/SIP-mediated G1-S shortening in human colon cancer cells after gastrin stimulation.Pretreatment with MG132, the 26S proteasome inhibitor, blocked the CacyBP/SIP-induced reduction of P27^kip1, suggesting the involvement of the 26S proteasome in the degradation of P27^kip1. To date, it has been reported that P27^kip1 seems to be the primary target of the SCFcomplex. Skp1 is the adaptor protein of SCF complex. Recently, based on the domain mapping studies, it has been confirmed that CacyBP/SIP's C-terminal region is responsible for interaction with Skp1. We also confirmed that CacyBP/SIP could bind Skp1 in HT29/SW480 cells, which is consistent with the consecutive proteasomal degradation of P27^kip1. To preliminarily assess whether CacyBP/SIP increase the degradation of P27^kip1through interaction with Skp1, we constructed the truncated mutation of CacyBP/SIP (Δ73–228) and fused it into pEGFP/C1. Using transient transfect, this mutant could be translocated into nuclear after gastrin induction but failed to interact with Skp1 by co-immunoprecipitation using transfected SW480-CacyBP/SIPsi cells. At the same time, no change was detected in the protein level of P27^kip1 after gastrin induction.【Conclusion】In conclusion, these results suggested that CacyBP/SIP promotes proliferation of colon cancer cells through enhancing ubquitin-mediated degradation of P27^kip1.