SOXs In Human Prostate Cancer:Implication As Progression And Prognosis Factors

Background and objectives:Prostate cancer (PCa) is one of the most prevalent malignancies in men and the second most frequent cause of male cancer-related death. With the development of society and life expectancy, the incidence of PCa has been in a coutinous rise. It is a clinically heterogeneous-multifocal disease. Carcinogenesis and the mechanisms influencing the progression and prognosis of PCa is a multistep process, involving both genetic insults to epithelial cells and changes in epithelial-stromal interactions. The serum prostate specific antigen(PSA) is currently the most commonly used molecular biological markers for diagnosis of PCa. PSA screening can detect PCa early and a large number of PCa to be cured. However, PSA has a good sensitivity but poor specificity, there is a diagnostic gray zone. Prostatitis, benign prostatic hyperplasia, acute urinary retention may also lead to an elevated PSA levels, coupled with the incidence of PCa is slow and occult, so can not be solely the PSA to determine the formation and prognosis of PCa. Each PCa patient on androgen deprivation therapy(ADT) are not the same degree of sensivity. Although ADT is able to reduce serum PSA levels and delay the progress of PCa, but almost all patients after14-30months of ADT exist androgen resistance that make the development of androgen-dependent prostate cancer to androgen-independent prostate cancer(AIPC) or hormone-resistant prostate cancer(HRPC) and result in a relapse of PCa, finally. Once the development of AIPC or HRPC, most patients died18months later.In recent years, the SOX genes(SOXs) in the diagnosis and treatment of tumors have more and more attention. SOXs belong to the High Mobility Group(HMG) superfamily and encode transcription fators. There are more than20SOXs, it has been demonstrated that SOXs can regulate cell differentiation and proliferation, including sex determination, chondrogenesis, and nervous system development. Based on phylogenetic analysis of their HMG domains, SOXs can be separated into subgroup A-J. SOXs have similar functions in the same subgroup, different subgroup SOXs can contact each other, and the functions of the same gene in the same cells in different developmental processes are not the same. SOXs as an oncogene, direct evidence is the ectopic SOX3expression of chick embryo fibroblasts can cause cancer which is regulated by HMG domain and transcriptional activation. SOXs also as an tumor antigen, for example, the small cell lung cancer patients that accompanied or not accompanied by myasthenia gravis syndrome(Lambert-Eaton myasthenic syndrome) often be detected SOX1antibodies, and can be used as serological markers for tumor detection. SOX10has also been identified as a tumor antigen in melanoma patients. The performance of the same SOXs in different tumors are different. For example, SOX7is upregulated in pancreatic cancer, gastric cancer and esophageal cancer cells, but significantly downregulated in primary colorectal cancer, breast cancer, renal cancer, lung cancer and PCa.With the developmental research of SOXs, SOXs and PCa relationship has become a hot research topic. Guo et al who specializes in the SOX7protein expression in PCa, found SOX7downregulated in47%of PCa, this may be a result of tumor-specific promoter hypermethylation. SOX7may reduce the activation of catenin to inhibit catenin-mediated transcription process, suggesting that SOX7is an independent checkpoint in prostate cells. SOX9is a more research and more thorough gene in SOXs. Drivdahl et al found that increased level of SOX9resulted in decreased rate of cell differentiation, cell cycle stopped at the G0/G1phase and increased apoptotic sensitivity. Thomsen et al found that SOX9expressed in the primary stage of prostate development in mice, the SOX9knockout lead to abnormal differentiation of ventral prostate developmental disorders, finally confirmed that SOX9was the essential gene for early differentiation of prostatic bud epithelium. Wang et al confirmed that SOX9expressed in primary PCa, and have a higher expression of SOX9in biochemical recurrence of PCa and PCa cell lines. SOX9is regulated by Wnt/beta-catenin pathway and play a role in the promotion of prostate development and maintenance prostate epithelial cells, and then they found that SOX9promote the differentiation of tumor cells in the PCa cell lines, that it may be associated with invasive growth of PCa. The latest research reports, SOX9is highly expressed at early tumor stage in Pten and NKx3.1mutant mice, it may be associated with all stages of tumor progression. In transgenic mice, the highly expression of SOX9promoted cell differentiation rather than proliferation, and that can rapidly induce well-differentiated prostate epithelial tumor, on the contrary, the low expression of SOX9reduced the proliferation of prostate tumor cells. Finally,880cases of PCa tissues by immunohistochemical experiment confirmed that the highly expression of SOX9was positive related with Gleason score. There was few studies about SOX10an PCa. Reported in the literature is mainly related to human melanoma. Zhong et al who found that SOX10in PCa tissues showed low expression, and it is positively correlated with SOX7, but negative with SOX9.There is evidence that androgen levels is the key factor for PCa recurrence, and the role of androgen receptor(AR) activation will be continued in spire of androgen levels in castration levels. The highly expression of AR will shorten the time of an elevated PSA after castration with ADT and AR nuclear stained positive patients have an increased mortality rate. Wang et al found that a high level of exogenous SOX9reduce the expression and activity of AR, on the contrary, low level of exogenous of SOX9promote the expression and activity of AR, SOX9regulated the expression of AR in PCa. The low expression of SOX10lead to a decreased expression of ErbB3, and inhibition of ErbB3can increase the sensitivity of PCa cells to androgen after ADT. SOX10may promote AR activity through inhibition the exression of ErbB3, and ultimately enhance the sensitivity of PCa cells to androgen.In order to further study the role of SOXs in diagnosis and determine recurrence in PCa, we used the preliminary gene chip screening and public database query, and select SOX7,SOX9and SOX10for studying. Detected their expression in PCa cell lines PC3, DU145, LNCap and immortalized prostate cell line RWPE-1by RT-QPCR, immunohischemical validation, combined with SOX7,SOX9and SOX10staining score and clinic pathological parameters were analyzed. All patients did not undergo castration and chemotherapy or radiation therapy before drawn, did not undergo prostate digital rectal examination and acute urinary one week before drawn, did not have history of prostatitis.Materials:147cases of PCa tissues and28cases of BPH tissues were obtained immediately during the operation of transurethral resection prostate and suprapubic prostatectomy from2001to2011Guangzhou First Municipal People’s Hospital, and19in147cases had been confirmed local or distant metastasis. Quick-frozen for collection and preservation. All the specimens were confirmed by two pathologists.PCa cell lines PC3, DU145, LNCap and immortalized prostate cell line RWPE-1are kept by Guangzhou First Municipal People’s Hospital Central Laboratory. Cell growth in good condition for the experiment.Methods:Using the gene chip to conduct a preliminary screening, and select SOX7,SOX9and SOX10that differentially expressed in PCa tissues and BPH tissues. Oncomine database query their expression.RT-QPCR detected the expression of SOX7, SOX9and SOX10in PC3, DU145, LNCap and RWPE-1. Using Trizol to extraction cell lines RNA, doing reverse transcription according to the manial operation of the TAKARA kit. Reaction using the primers of these genes in quantitative PCR instrument, the reaction parameters:95℃for5minutes, then95℃for10seconds,60℃for20seconds,72℃for20seconds, a total of40cycles. The IQ5software output the results after the completion of the reaction, the expression of each gene was standardized by the beta-actin expression values, using the comparative CT value method to analyze the differentially expression. All the specimens were fixed in10%neutral buffered formalin and subsequently embedded in paraffin, cut into slices of thick4um. Hydrogen peroxide labeled streptomycin avidin-hydrogen peroxide enzymatic(SP) immunohistochemical(IHC) staining. A final immunoreactivity scores(IRS) was obtained for each case by multiplying the percentage and the intensity score, with PBS instead of primary antibody as a negative control. Protein expression levels were further analyzed by classifying IRS values as low(based on a IRS value less than4, negative) and as high(based on a IRS value greater than4, positive). The IRS was done by two pathologists.Statistical treatment:The software of SPSS version13.0for windows was used for statistical analysis. Continuous variables were expressed as χ±s. One-way ANOVA test was performed for analyzing the expression of SOX7,SOX9and SOX10in cell lines, pairwise comparisons using the Bonferroni method, Pearson χ2test for their staining score in PCa and BPH tissues. Pearson correlation was calculated between the expression levels of SOX7, SOX9and SOX10in PCa tissues. Independent-samples T test analysis the relationship between IRS and clinical pathological parameters. Kaplan-Meier method for the question of survival, and Cox regression analysis for the multivariate analysis. Differences were considered statistically significant when P was less than0.05.Results:1. Our gene chip screening showed that, relative to BPH tissues, SOX7(P=0.001) and SOX10(P=0.010) is down-regulated in PCa tissues, while SOX9(P=0.029) is up-regulated in PCa tissues.2. Query SOX7, SOX9and SOX10expression in PCa tissue and noncancerous tissue through Oncomine public database, we found that SOX7and SOX10is down-regulated in PCa tissues, while SOX9showed up-regulation.3. We detected the expression of SOX7,SOX9and SOX10in PCa cell lines and immortalized prostate cell line RWPE-1, we found that the expression of SOX9in PC3cell line was significantly higher than that in RWPE-1cell line(P=0.026). We also found that the expression of SOX10in PC3was significantly lower than that in RWPE-1(P=0.001), but in LNCap cell lines was significantly higher than that in RWPE-1(P=0.001), and there was not differentially expression of SOX7was observed in the above cell lines.4. SOX7and SOX9expression occurred mainly in the nucleus, while SOX10in the cytoplasm. Related to BPH tissues, SOX7(x2=5.041,P=0.025) and SOX10(χ2=6.703,P=0.010) was low expressed in PCa tissues, and SOX9(x2=4.105,P=0.043) was highly expression in PCa tissues.5. The expression level of SOX7protein in PCa tissues was significantly negative correlated with that of SOX9protein(r=-0.263,P=0.001) and positive correlated with that of SOX10protein(r=0.422,P=0.000), respectively. The expression level of SOX9protein was also significantly negative correlated with that of SOX10protein(r=-0.377,P=0.000).6. The combination of IHC staining scores and corresponding clinicopathological features of PCa patients were analyzed, we found that the staining scores of SOX7in PCa patients in serum PSA levels less than10ng/ml(t=1.994,P=0.048),Gleason score less than7points(t=-2.034,P=0.047), clinical stage less than T2A(t=4.245,P=0.000) and pathological stage in T2A-T2C(t=3.224,P=0.002) had a higher expression. There was a higher expression of SOX9in PCa patients with Gleason score which greater than or equal to7points(t=3.393,P=0.001) and clinical stage greater than T2A(t2.871,P=0.005). The expression of SOX10was higher in Gleason score less than7points(t=-2.902,P=0.006) and lower in clinical stage greater than or equal to T2A(t=3.365,P=0.001). The expression of S0X7in PCa was negative related with malignancy, and the expression of SOX9was positive related with malignancy. It was unable to determine the exact relationship of SOX10expression in PCa and malignancy.7. Staining score greater than or equal to4(posive) and less than4(negative) PCa tissues were divided into high expression group and the low expression group. The association of the expression levels of SOX7, SOX9and SOX10with the biochemical recurrence-free survival, overall survival and metastasis-free survival of PCa patients was analyzed using Kaplan-Meier method. The data indicated that there were no significant difference in over and metastasis-free survival between high expression group and low expression group of SOX7(all P>0.05). Interestingly, the results by pairwise comparisons showed that there is a significant difference in the biochemical recurrence-free survival rates between patients with high SOX7expression and low SOX7expression(Log Rank=10.790,P=0.001). In addition, the biochemical recurrence-free survival, overall survival and metastasis-free survival of patients with high SOX9expression were significantly lower than those with low SOX9expression(Log Rank=7.224, P=0.007; Log Rank=10.912,P=0.001; Log Rank=4.301,P=0.038), respectively. There was no significantly correlation observed with SOX10and biochemical recurrence-free survival, overall survival and metastasis-free survival.8. By the above method, PCa tissues were divided into the high expression group and the low exression group. The multivariate analyses showed that SOX7(RR=0.300, P=0.003) and SOX9(RR=0.121, P<0.001) were protected factors of biochemical recurrence-free survival, and the highly expression of SOX7and SOX9had a better biochemical recurrence-free survival.Conclusions:1. We used the gene chip to screen SOXs which differentially expressed in PCa tissues and BPH tissues, and then IHC validation, that indicated that SOX7, SOX10may be tumor suppressor genes, SOX9is a cancer-promoting gene. It may be identify PCa and BPH through detecting the expression levels of SOX7,SOX9and SOX10.2. SOX7/SOX9and SOX9/SOX10combination of genes may be diagnostic of PCa. It may be improve the efficiency of the early diagnosis PCa by detecting the two gene combinations.3. The combination of IHC staining scores and corresponding clinicopathological features of PCa patients were analyzed, we found that the de-regulation of SOX7and SOX9was associated with the progress of PCa, and it may determine the degree of malignancy of PCa by detecting the levels of these genes in vivo.4. Combination of SOX7and SOX9in the relationship in PCa, we established the mechanism of SOX7and SOX9bi-directional regulation for the further study of PCa.5. The highly expression of SOX7and SOX9were significantly associated with biochemical recurrence-free survival, and subsequent the multivariate analyses showed that the up-regulation of SOX7SOX9were protected factors of biochemical recurrence-free survival, and they had a better biochemical recurrence-free survival. It may predict the early risk of biochemical recurrence and judge the prognosis of PCa by detecting SOX7, SOX9levels.6. The data indicated that a high level of SOX7and SOX9may inhibit biochemical recurrence of PCa by inhibiting the expression of AR level. SOX9plays a dual role in the development of PCa, it is a cancer-promoting gene, sometimes, it turns a tumor suppressor gene.