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The clinical and biological significance of STAT1 in esophageal squamous cell carcinoma.

Zhang Y, Molavi O, Su M, Lai R - BMC Cancer (2014)

Bottom Line: Furthermore, patients with STAT1-strong/weak tumors had a significantly longer survival compared to those with STAT1-negative tumors (33.6 months versus 13.1 months, p=0.019).In patients carrying tumors of aggressive cytology (n=50), those with STAT1-strong tumors survived significantly longer than those with STAT1-weak/negative tumors (34.6 months versus 20.5 months, p=0.011).To conclude, our findings suggest that STAT1 is a tumor suppressor in ESCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Shantou University Medical College, 22 Xinling Road, Shantou 515031, Guangdong Province, China. minsu@stu.edu.cn.

ABSTRACT

Background: Loss of STAT1 (Signal Transducer and Activator of Transcription-1) has been implicated in the pathobiology of a number of cancer types. Nonetheless, the biological and clinical significance of STAT1 in esophageal squamous cell carcinomas (ESCC) has not been comprehensively studied.

Methods: Using immunohistochemistry, we detected the STAT1 expression in a cohort of ESCC patients; In-vitro experiments, we used enforced gene transfection of STAT1C into two STAT1- weak/negative ESCC cell lines and siRNA knockdown of STAT1 in two STAT1-strong ESCC cell lines to detect STAT1 function in ESCC.

Results: We found that the expression of STAT1 was heterogeneous in ESCC, with 64 (49.0%) strongly positive cases, 59 (45.0%) weakly positive cases and 8 (6.1%) negative cases. STAT1 expression inversely correlated with the depth of tumor invasion and tumor size (p=0.047 and p=0.029, respectively, Chi square). Furthermore, patients with STAT1-strong/weak tumors had a significantly longer survival compared to those with STAT1-negative tumors (33.6 months versus 13.1 months, p=0.019). In patients carrying tumors of aggressive cytology (n=50), those with STAT1-strong tumors survived significantly longer than those with STAT1-weak/negative tumors (34.6 months versus 20.5 months, p=0.011). Our in-vitro experiments revealed that STAT1 is proapoptotic and inhibitory to cell-cycle progression and colony formation. Lastly, we found evidence that STAT1 signaling in ESCC cells down-regulated the expression and/or activity of NF-κB and STAT3, both of which are known to have oncogenic potential.

Conclusion: To conclude, our findings suggest that STAT1 is a tumor suppressor in ESCC. Loss of STAT1, which is frequent in ESCC, contributes to the pathogenesis of these tumors.

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Gene transfection ofSTAT1Cupregulated apoptosis and induced sub-G1cell cycle increase. By western blots, gene transfection of STAT1C into ESCC cell lines induced cleavages of caspase 3, downregulated several pro-apoptotic proteins (including BCL-2, BCL-xL, survivin), and promoted G1 cell-cycle arrest by decreasing cyclin D1 and increasing p21waf1. Cell lysates were collected 2 days after the gene transfection of STAT1C in EC1 and EC109 (A). Time course experiments were performed, and the decrease in cyclin D1 expression was detectable as early as 6 hours after STAT1C transfection in EC1 cells (B). (C) Cell cycle analysis using flow cytometry revealed that STAT1C induced a significant increase in the sub-G1 fraction in both cell lines, EC1 and EC109 (*p < 0.05). All experiments were performed in triplicate, and results from a representative run are shown. (E.V.: empty vector).
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Fig4: Gene transfection ofSTAT1Cupregulated apoptosis and induced sub-G1cell cycle increase. By western blots, gene transfection of STAT1C into ESCC cell lines induced cleavages of caspase 3, downregulated several pro-apoptotic proteins (including BCL-2, BCL-xL, survivin), and promoted G1 cell-cycle arrest by decreasing cyclin D1 and increasing p21waf1. Cell lysates were collected 2 days after the gene transfection of STAT1C in EC1 and EC109 (A). Time course experiments were performed, and the decrease in cyclin D1 expression was detectable as early as 6 hours after STAT1C transfection in EC1 cells (B). (C) Cell cycle analysis using flow cytometry revealed that STAT1C induced a significant increase in the sub-G1 fraction in both cell lines, EC1 and EC109 (*p < 0.05). All experiments were performed in triplicate, and results from a representative run are shown. (E.V.: empty vector).

Mentions: Using the ESCC cell lines, we then performed specific in-vitro studies. First, we examined the biological impact of enforced expression of the constitutively active form of STAT1 (i.e. STAT1C) in ESCC. To this end, EC1 and EC109, both of which were STAT1-weak/negative cell lines, were employed and they were subjected to gene transfection of STAT1C. As shown in Figure 3A, the expression of STAT1C was confirmed by the high intensity of the total STAT1 band and the strong expression of FLAG, which was tagged to the STAT1C construct. These changes correlated with a significant decrease in the number of viable cells, as assessed using the trypan blue exclusion assay (Figure 3B). As shown in Figure 3C and D, STAT1C transfection in EC1 and EC109 cells led to a significant decrease in colony formation and cell invasion, as compared to cells transfected with the empty vector (p < 0.001 and p < 0.05 in both cell lines). As shown in Figure 4A, the occurrence of apoptosis was supported by the expression of cleaved caspase 3 in both cell lines. Correlating with these changes, there was a marked reduction in the expression levels of several anti-apoptotic proteins including BCL-2, BCL-xL and survivin. Furthermore, we also observed changes in two proteins known to regulate G1 cell-cycle progression including p21Waf1 and cyclin D1. Specifically, transfection of STAT1C into EC1 and EC109 substantially upregulated p21Waf1, a negative regulator of G1 cell-cycle progression [14]. Cyclin D1, a promoter of G1 cell-cycle progression [15], was downregulated. Based on the results of the time-course experiment (Figure 4B), the decrease in the cyclin D1 protein level began as early as 6 hours after STAT1C gene transfection, indicating that the decrease in cyclin D1 was not due to the apoptotic activity. As shown in Figure 4C, cell cycle analysis showed a significant increase in the sub-G1 fractions in EC1 and EC109 cells transfected with STAT1C.Figure 3


The clinical and biological significance of STAT1 in esophageal squamous cell carcinoma.

Zhang Y, Molavi O, Su M, Lai R - BMC Cancer (2014)

Gene transfection ofSTAT1Cupregulated apoptosis and induced sub-G1cell cycle increase. By western blots, gene transfection of STAT1C into ESCC cell lines induced cleavages of caspase 3, downregulated several pro-apoptotic proteins (including BCL-2, BCL-xL, survivin), and promoted G1 cell-cycle arrest by decreasing cyclin D1 and increasing p21waf1. Cell lysates were collected 2 days after the gene transfection of STAT1C in EC1 and EC109 (A). Time course experiments were performed, and the decrease in cyclin D1 expression was detectable as early as 6 hours after STAT1C transfection in EC1 cells (B). (C) Cell cycle analysis using flow cytometry revealed that STAT1C induced a significant increase in the sub-G1 fraction in both cell lines, EC1 and EC109 (*p < 0.05). All experiments were performed in triplicate, and results from a representative run are shown. (E.V.: empty vector).
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Related In: Results  -  Collection

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Fig4: Gene transfection ofSTAT1Cupregulated apoptosis and induced sub-G1cell cycle increase. By western blots, gene transfection of STAT1C into ESCC cell lines induced cleavages of caspase 3, downregulated several pro-apoptotic proteins (including BCL-2, BCL-xL, survivin), and promoted G1 cell-cycle arrest by decreasing cyclin D1 and increasing p21waf1. Cell lysates were collected 2 days after the gene transfection of STAT1C in EC1 and EC109 (A). Time course experiments were performed, and the decrease in cyclin D1 expression was detectable as early as 6 hours after STAT1C transfection in EC1 cells (B). (C) Cell cycle analysis using flow cytometry revealed that STAT1C induced a significant increase in the sub-G1 fraction in both cell lines, EC1 and EC109 (*p < 0.05). All experiments were performed in triplicate, and results from a representative run are shown. (E.V.: empty vector).
Mentions: Using the ESCC cell lines, we then performed specific in-vitro studies. First, we examined the biological impact of enforced expression of the constitutively active form of STAT1 (i.e. STAT1C) in ESCC. To this end, EC1 and EC109, both of which were STAT1-weak/negative cell lines, were employed and they were subjected to gene transfection of STAT1C. As shown in Figure 3A, the expression of STAT1C was confirmed by the high intensity of the total STAT1 band and the strong expression of FLAG, which was tagged to the STAT1C construct. These changes correlated with a significant decrease in the number of viable cells, as assessed using the trypan blue exclusion assay (Figure 3B). As shown in Figure 3C and D, STAT1C transfection in EC1 and EC109 cells led to a significant decrease in colony formation and cell invasion, as compared to cells transfected with the empty vector (p < 0.001 and p < 0.05 in both cell lines). As shown in Figure 4A, the occurrence of apoptosis was supported by the expression of cleaved caspase 3 in both cell lines. Correlating with these changes, there was a marked reduction in the expression levels of several anti-apoptotic proteins including BCL-2, BCL-xL and survivin. Furthermore, we also observed changes in two proteins known to regulate G1 cell-cycle progression including p21Waf1 and cyclin D1. Specifically, transfection of STAT1C into EC1 and EC109 substantially upregulated p21Waf1, a negative regulator of G1 cell-cycle progression [14]. Cyclin D1, a promoter of G1 cell-cycle progression [15], was downregulated. Based on the results of the time-course experiment (Figure 4B), the decrease in the cyclin D1 protein level began as early as 6 hours after STAT1C gene transfection, indicating that the decrease in cyclin D1 was not due to the apoptotic activity. As shown in Figure 4C, cell cycle analysis showed a significant increase in the sub-G1 fractions in EC1 and EC109 cells transfected with STAT1C.Figure 3

Bottom Line: Furthermore, patients with STAT1-strong/weak tumors had a significantly longer survival compared to those with STAT1-negative tumors (33.6 months versus 13.1 months, p=0.019).In patients carrying tumors of aggressive cytology (n=50), those with STAT1-strong tumors survived significantly longer than those with STAT1-weak/negative tumors (34.6 months versus 20.5 months, p=0.011).To conclude, our findings suggest that STAT1 is a tumor suppressor in ESCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Shantou University Medical College, 22 Xinling Road, Shantou 515031, Guangdong Province, China. minsu@stu.edu.cn.

ABSTRACT

Background: Loss of STAT1 (Signal Transducer and Activator of Transcription-1) has been implicated in the pathobiology of a number of cancer types. Nonetheless, the biological and clinical significance of STAT1 in esophageal squamous cell carcinomas (ESCC) has not been comprehensively studied.

Methods: Using immunohistochemistry, we detected the STAT1 expression in a cohort of ESCC patients; In-vitro experiments, we used enforced gene transfection of STAT1C into two STAT1- weak/negative ESCC cell lines and siRNA knockdown of STAT1 in two STAT1-strong ESCC cell lines to detect STAT1 function in ESCC.

Results: We found that the expression of STAT1 was heterogeneous in ESCC, with 64 (49.0%) strongly positive cases, 59 (45.0%) weakly positive cases and 8 (6.1%) negative cases. STAT1 expression inversely correlated with the depth of tumor invasion and tumor size (p=0.047 and p=0.029, respectively, Chi square). Furthermore, patients with STAT1-strong/weak tumors had a significantly longer survival compared to those with STAT1-negative tumors (33.6 months versus 13.1 months, p=0.019). In patients carrying tumors of aggressive cytology (n=50), those with STAT1-strong tumors survived significantly longer than those with STAT1-weak/negative tumors (34.6 months versus 20.5 months, p=0.011). Our in-vitro experiments revealed that STAT1 is proapoptotic and inhibitory to cell-cycle progression and colony formation. Lastly, we found evidence that STAT1 signaling in ESCC cells down-regulated the expression and/or activity of NF-κB and STAT3, both of which are known to have oncogenic potential.

Conclusion: To conclude, our findings suggest that STAT1 is a tumor suppressor in ESCC. Loss of STAT1, which is frequent in ESCC, contributes to the pathogenesis of these tumors.

Show MeSH
Related in: MedlinePlus