Clinicopathological Significance Of SATB1 And SATB2 In Colorectal Cancer And The Mechanisms Underlying Their Effects On EMT

BACKGROUND AND OBJECTIVEColorectal cancer is one of the most common tumors in modern society,and incidence rates of CRC continue to increase in economically transitioning countries Although various modifiable risk factors for CRC have been found,the molecular mechanisms remain poorly understood.Therefore,a more comprehensive understanding about the genetic and epigenetic alterations that drive the development and progression of CRC is sorely required.SATB family,including SATB1 and SATB2,nuclear matrix attachment region binding protein that acts as a transcription factor and epigenetic regulator by forming chromatin loci and recruiting histone-modified enzymes.Early studies suggest that SATB 1 and SATB2 play indispensable roles in the development of organisms,especially in the central nervous system and bone differentiation.Recently,it has been found that SATB expression appears to be tissue-specific and plays an important role in the growth and metastasis of tumors.EMT is a classic change in the process of tumor progression and is also present in CRC.It is known that ?-catenin is a key molecule of the Wnt pathway in colorectal cancer.Interestingly,SATB1 can form complexes with ?-catenin in T cells and regulate many downstream target genes.Whether SATB1 can induce EMT through ?-catenin pathway need to be explored.Therefore,the aim of this study is to investigate the expression of SATB1 and SATB2 in CRC tissues and metastatic lymph nodes,and the relationship between SATB1/2 expression pattern and clinicopathological variables.In addition,we analyse the correlation between SATB1 expression and EMT,and explored the probable mechanism of SATB1 action in CRC.At the same time,due to the tissue specificity of SATB2 expression in the lower digestive tract,we also investigate clinical diagnostic value of SATB2 in CRC combined with CK20 and CK7.MATERIALS AND METHODSA total of 200 patients with colorectal cancer underwent radical resection from January 2011 to June 2013 were collected,including 50 cases of normal colorectal mucosa,200 cases of colorectal cancer primary cancer tissues,and 80 cases of matched lymph node metastases,5 cases of distant metastatic cancer tissue.And all the clinical data was collected and follow-up.The expression levels and characteristics of SATB1 and SATB2 proteins in the specimens were detected by tissue microarray and immunohistochemistry.The correlations between SATB1,SATB2 and clinicopathological features or prognosis were further analysed.The expression of SATB2,CK20 and CK7 protein in 200 cases of colorectal cancer primary carcinoma,135 cases of non-colorectal cancer and 9 cases of distant carcinoma of colorectal cancer were detected by immunohistochemistry.Sensitivity and specificity of each indicator was calculated alone and in combination seperately.The expression pattern of ?-catenin in CRC and the expression of E-cadherin,CK20,Vimentin,SMA and desmin were detected by immunohistochemistry.The correlation between SATB1 expression and aberrant localisation of ?-catenin and the expression of EMT markers were furtherly analyzed.The co-localization of SATB1 protein and ?-catenin protein in human CRC tissues was observed by immunofluorescence double staining.Furthermore,we overexpreessed SATB1 in colorectal cancer cell lines SW620.Immunoprecipitation method revealed that SATB1 and ?-catenin can combine to form a protein complexe.Western blot was used to detect the expression and location changes of(3-catenin protein and EMT related genes like Snail,Slug and TwistRESULTS1.Correlation of clinicopathological features with expression of SATB1 protein in CRCThe expression rate of SATB1 in CRC was 66.5%(133/200),which was significantly higher than that in normal mucosa(28%,14/50).The expression rate of SATB1 in lymph node metastases was 75%(60/80),significantly higher than the previous two groups(P=0.000).The high expression level of SATB1 in primary lesions and lymph node metastases was closely related to the low differentiation of CRC and had no correlation with other pathological factors.Multivariate analysis showed that the factors which can affect the OS rate of CRC patients were:poor differentiation,deep infiltration,distant metastasis and right colon,but not with SATB1 expression2.The mechanism of SATB1 in the process of EMT in CRCHigh expression of SATB1 was significantly correlated with aberrant expression of ?-catenin(P=0.0005),low expression of E-cadherin(P=0.000)and CK20(P=0.000)and with high expression of Vimentin(P=0.001).No SMA or desmin protein was expressed in the CRC cells.Overexpression of SATB1 in colorectal cancer cell line SW620 increases the translocation to nucleus of ?-catenin,and then form a protein complex to increase the expression of downstream genes,such as Snail,Slug and Twist.At the same time,the cell morphology changes from epithelial morphology to interstitial cell morphology.3.Correlation of clinicopathological features with expression of SATB2 protein in CRCThe expression rate of SATB2 in primary CRC was 61.0%(122/200),which was lower than that in normal intestinal mucosa(82%,41/50).The expression rate of lymph node metastasis was 43.8%(35/80),which was significantly lower than that of the former two groups(P=0.000).The expression of SATB2 in primary colorectal cancer was significantly correlated with tumor size,differentiation,depth of invasion,lymph node metastasis and TNM staging(P<0.05),and was not related to age and sex.Multivariate analysis showed that the high expression level of SATB2 was a good predictor of the prognosis of patients with CRC(HR=0.685,P=0.012).4.Diagnostic value of SATB2 as a CRC markerThe sensibility and specificity were 80.5%and 92%respectively when CK20+/CK7-phenotype was used in the diagnosis of colorectal cancer.When a triple combination of markers was analyzed,the phenotype CK20+/CK7-/SATB2+had a sensitivity of 59%and a specificity of 98%.For the combination SATB2+/CK7-or CK20+/CK7-,the sensitivity was 88%and specificity was 90%.There was only one case showed weakly expression of SATB2 in primary ovarian tumors(4.4%,1/23),and the expression of SATB2 was negative in all 5 cases of myxoid tumors.Four cases of CRC distant metastatic carcinoma with CK20-/CK7-phenotypic showed SATB2 positive expression.Three cases of appendix mucinous adenocarcinoma all showed explicitly SATB2 expression.CONCLUSION1.SATB1 plays as an oncogene in CRC,and its expression in tumor is significantly higher than that in normal tissue,and its high expression is closely related to poor differentiation of CRC.2.SATB1 might promote the epithelial-mesenchymal transition by increasing the aberrant expression of ?-catenin,and combining with it to form a protein complex,increasing the transcription of downstream EMT regulators,like Snail,Slug and Twist,and than promote the EMT process.3.SATB2 may play a role in CRC as a suppressor gene.Low expression of SATB2 was associated with the development and progression of colorectal cancer.And its expression is significantly decreased during tumor metastasis.Furthermore,SATB2 expression is closely related to poor prognosis.4.Combined detection of CK20+/CK7-/SATB2+can increase the specificityof CRC diagnosis significantly,particularly in the differential diagnosis of CRC metastases and primary ovarian tumors.SATB2 can be used as an important complementary immunohistochemical marker in diagnosis of colorectal cancer SATB2 was also a highly sensitive marker for the detection of appendiceal tumors.