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

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.

Figure 1

Heterogeneous STAT1 expression in ESCC. (A) By immunohistochemistry applied to formalin-fixed paraffin-embedded tissues, variable levels of STAT1 were detectable in most ESCC tumors examined. The staining was predominantly cytoplasmic. Based on the staining intensity, tumors in our cohort was categorized into STAT1-strong (a) or STAT1-weak (b); 8 cases were STAT1-negative (c) (IHC stain, scale bar, 20 μm). Nuclear staining of STAT1 was detected in some ESCC cases (d) (IHC stain, scale bar, 50 μm). The normal epithelium (e) from a STAT1-weak tumor (shown in f) was also illustrated (scale bar, 20 μm). (B) By Western blots, STAT1 expression in ESCC tumors was examined. Compared to the benign esophageal tissue harvested at the surgical margins in the same specimens (labeled as N) cancerous tissues (labeled as Ca) often expressed a lower level of STAT1. Thus, tumors from patient #1, 2 and 4 were categorized as STAT1-low. A small subset of tumors (e.g. that from patient #3) were categorized as STAT1-high, since the expression of STAT1 in the cancerous tissue was appreciably higher than that of the benign esophageal tissues in the same specimen. (C) By Kaplan-Meier analysis, we found no significant correlation between overall survival and the expression level of STAT1, when the two groups were defined as STAT1-strong and STAT1-weak/negative (a). In contrast, we found a significant correlation between overall survival and the expression level of STAT1 protein levels when the two groups were defined as STAT1-positive or STAT1-negative (b). With the subset of patients carrying poorly or intermediate-differentiated tumors, those with STAT1-strong tumors survived significantly longer than those with STAT1-weak/negative tumors (c).

Figure 2

Expression of STAT1 and phospho-STAT1 in ESCC (n = 4) and esophageal immortalized cell lines (n = 4). ESCC cell lines included EC1, EC109, KYESE150 and KYSE510 and human esophageal immortalized cell lines included SHEE, NE2, NE3 and NE6. MCF7, a breast cancer cell line, served as a positive control. The expression of STAT1 was heterogeneous among these cell lines, and the expression of phospho-STAT1 was generally in parallel with the expression of STAT1.

Figure 3

Gene transfection of STAT1C significantly decreases cell growth and tumorigenecity in ESCC cell lines. Using Western blot analysis, the gene transfection of STAT1C in EC1 and EC109 cells was shown to be effective, since the levels of STAT1, phospho-STAT1 and FLAG were dramatically increased 2 days after STAT1C transfection (A). Cell growth, as assessed by trypan blue cell counting, was found to be significantly decreased after STAT1C transfection in EC1 and EC109 cells (B) (* p < 0.05). Tumorigenecity, assessed by using colony formation assay, was significantly lower in EC1 and EC109 cells transfected with STAT1C, as compared to cells transfected with an empty vector (C) (** p < 0.001). (D) Transwell invasion assay showed that the transfection of STAT1C significantly inhibited cell invasion both ESCC cell lines. Results shown are representative of three independent experiments. (E.V.: empty vector).

Figure 4

Gene transfection of STAT1C upregulated apoptosis and induced sub-G ₁ cell 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 p21^waf1. 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).

Figure 5

Inhibition of STAT1 activation by siRNA. By Western blot analysis, the protein level of STAT1 and phospho-STAT1 were dramatically decreased in KYSE150 and KYSE510 treated with siRNA against STAT1. Cell lysates were collected 2 days after the siRNA transfection (A). The decrease in STAT1 expression after siRNA treatment was further supported by quantitative RT-PCR (***p < 0.0001) (B). In both KYSE150 and KYSE510, siRNA knockdown of STAT1 induced a significant decrease in cell growth, assessed by trypan blue eclusion assay. The cell numbers were assessed on day 4 after siRNA transfection. Triplicate experiments were performed and the results of a representative experiment are illustrated (*p < 0.05) (C). Transfection of STAT1 siRNA into KYSE150 and KYSE510 cells led to a significant reduction in the number of colonies formed, as compared to cells transfected with scrambled siRNA. Triplicate experiments were performed and the results of a representative experiment are shown (***p < 0.0001) (D). By western blots, transfection of STAT1 siRNA resulted in an appreciable increase in BCL-xL, BCL-2, cyclin D1 and a corresponding decrease in p21^waf1. Cells treated with scrambled siRNA served as the negative controls. Cell lysates were prepared two days after siRNA transfection (E). Cell cycle analysis using flow cytometry revealed that STAT1 siRNA induced a significant decrease in the sub-G1 fraction in both cell lines, KYSE150 and KYSE510 (*p < 0.05). Results shown are representative of three independent experiments (F).

Figure 6

STAT1C inhibits NF-κB signaling. Western blot results showed a detectable down-regulation of total p65 and phospho-p65 after STAT1C transfection in EC1 and EC109 cells (A). In the same experiment, nuclear/cytoplasmic fractionation studies showed that STAT1C induced a substantial decrease in nuclear p65 and phospho-p65 (B). (C) Using a NF-κB/luciferase reporter, we found that STAT1C gene transfection induce a significant down-regulation of the NF-κB transcription activity in ESCC cells transfected with STAT1C cells were harvested 48 hours after the gene transfection. (*p < 0.05) (E.V.: empty vector).

Figure 7

STAT1C decreases the expression level of STAT3 and phospho-STAT3 and increase STAT1-STAT3 heterodimer formation. Western blot studies showed that gene transfection of STAT1C induced an appreciable decrease in total STAT3 and phospho-STAT3 in EC1 and EC109 cells. Cell lysates were harvested 48 hours after gene transfection (A). In contrast, both STAT3 and phospho-STAT3 were increased in response to siRNA knockdown of STAT1 in KYSE150 and KYSE510 (B). Co-immunoprecipitation experiments revealed that gene transfection of STAT1C into EC1 and EC109 induced a substantial increase in STAT1:STAT3 heterodimer; at the same time, the total STAT3 protein level was decreased (C). Cell lysates were prepared 48 hours after gene transfection. (E.V.: empty vector).