Apollon modulates chemosensitivity in human esophageal squamous cell carcinoma

Patients with esophageal squamous cell carcinoma (ESCC) are often diagnosed with advanced diseases that respond poorly to chemotherapy. Here we reported that Apollon, a membrane-associated inhibitor of apoptosis protein, was overexpressed in ESCC cell lines and clinical ESCC tissues, and Apollon overexpression clinically correlated with poor response to chemotherapy (P = 0.001), and short overall survival (P = 0.021). Apollon knockdown increased cisplatin/docetaxel-induced apoptosis, mitochondrial dysfunction and cytochrome c release in two ESCC cell lines. Apollon knockdown potentiated cisplatin/docetaxel-induced long-term cell growth inhibition, and enhanced chemosensitivity of ESCC cells to cisplatin/docetaxel in xenograft tumor models. Apollon knockdown also enhanced cisplatin/docetaxel-induced activation of caspase-8 (extrinsic pathway) and caspase-9 (intrinsic pathway) in ESCC cells and xenograft tumor models. Mechanism studies revealed that the effect of Apollon on chemosensitivity is mainly mediated by Smac. Apollon expression strongly and negatively correlated with Smac expression in clinical ESCC tissues (P = 0.001). Apollon targeted Smac for degradation in ESCC cells. The effect of Apollon on chemosensitivity was reversed by Smac knockdown in ESCC cells. Taken together, our data show association of Apollon expression with chemotherapeutic response in ESCC, and provide a strong rationale for combining Apollon antagonism with chemotherapy to treat ESCC.     

High expression of long non‐coding RNA AFAP1‐AS1 predicts chemoradioresistance and poor prognosis in patients with esophageal squamous cell carcinoma treated with definitive chemoradiotherapy

To evaluate the clinical significance of lncRNAs in the resistance to cisplatin‐based chemoradiotherapy in esophageal squamous cell carcinoma (ESCC). We focused on lncRNAs which were frequently reported in ESCC or were involved in chemoradiotherapy resistance. LncRNA expressions were examined in paired cisplatin‐resistant and parental ESCC cell lines. Dysregulated lncRNAs were further measured in 162 pretreatment biopsy specimens of ESCC who received definitive chemoradiotherapy (dCRT). Then the correlations between lncRNA expression and response to dCRT and prognosis were analyzed. Three lncRNAs (AFAP1‐AS1, UCA1, HOTAIR) were found to be deregulated in cisplatin‐resistant cells compared with their parent cells. AFAP1‐AS1 was significantly up‐regulated in tumor tissues compared with adjacent normal tissues (P = 0.006). Furthermore, overexpression of AFAP1‐AS1 was closely associated with lymph node metastasis (P < 0.001), distant metastasis (P = 0.016), advanced clinical stage (P = 0.002), and response to dCRT (P < 0.001). Kaplan–Meier survival analysis revealed that high expression of AFAP1‐AS1 was significantly associated with shorter progression free survival (PFS) (median, 15 months vs. 27 months, P < 0.001) and overall survival (OS) (median, 29 months vs. 42 months, P < 0.001). In the multivariate analysis, high expression of AFAP1‐AS1 was found to be an independent risk factor to predict poor PFS (HR, 1.626; P = 0.027) and OS (HR, 1.888; P = 0.004). Thus, high expression of AFAP1‐AS1 could serve as a potential biomarker to predict tumor response and survival. Determination of this lncRNA expression might be useful for selection ESCC patients for dCRT. © 2016 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc.     

ASNA1, an ATPase targeting tail-anchored proteins, regulates melanoma cell growth and sensitivity to cisplatin and arsenite

Purpose  ASNA1 is homologous to E. coli ArsA, a well characterized ATPase involved in efflux of arsenite and antimonite. Cells resistant to arsenite and antimonite are cross-resistant to the chemotherapeutic drug cisplatin. ASNA1 is also an essential ATPase for the insertion of tail-anchored proteins into ER membranes and a novel regulator of insulin secretion. The aim of this study was to determine if altered ASNA1 levels influenced growth and sensitivity to arsenite and cisplatin in human melanoma cells. Methods  Cultured melanoma T289 cells were transfected with plasmids containing sense or antisense ASNA1. Cells were exposed to cisplatin, arsenite and zinc. Cell growth and chemosensitivity were evaluated by the MTT assay and apoptosis by a TUNEL assay. Results  ASNA1 expression was necessary for growth. T289 clones with decreased ASNA1 expression exhibited 51 ± 5% longer doubling times than wildtype T289 (P = 0.0091). After exposure to cisplatin, ASNA1 downregulated cells displayed a significant increase in apoptosis. The cisplatin IC₅₀ in ASNA1 underexpressing cells was 41.7 ± 1.8% compared to wildtype (P = 0.00097) and the arsenite IC₅₀ was 59.9 ± 3.2% of wildtype IC₅₀ (P = 0.0067). Conclusions  Reduced ASNA1 expression is associated with significant inhibition of cell growth, increased apoptosis and increased sensitivity to cisplatin and arsenite.     

Clinical significance of signal regulatory protein alpha (SIRPα) expression in esophageal squamous cell carcinoma

Signal regulatory protein alpha (SIRPα) is a type I transmembrane protein that inhibits macrophage phagocytosis of tumor cells upon interaction with CD47, and the CD47-SIRPα pathway acts as an immune checkpoint factor in cancers. This study aims to clarify the clinical significance of SIRPα expression in esophageal squamous cell carcinoma (ESCC). First, we assessed SIRPα expression using RNA sequencing data of 95 ESCC tissues from The Cancer Genome Atlas (TCGA) and immunohistochemical analytic data from our cohort of 131 patients with ESCC. Next, we investigated the correlation of SIRPα expression with clinicopathological factors, patient survival, infiltration of tumor immune cells, and expression of programmed cell death-ligand 1 (PD-L1). Overall survival was significantly poorer with high SIRPα expression than with low expression in both TCGA and our patient cohort (P < .001 and P = .027, respectively). High SIRPα expression was associated with greater depth of tumor invasion (P = .0017). Expression of SIRPα was also significantly correlated with the tumor infiltration of M1 macrophages, M2 macrophages, CD8⁺ T cells, and PD-L1 expression (P < .001, P < .001, P = .03, and P < .001, respectively). Moreover, patients with SIRPα/PD-L1 coexpression tended to have a worse prognosis than patients with expression of either protein alone or neither. Taken together, SIRPα indicates poor prognosis in ESCC, possibly through inhibiting macrophage phagocytosis of tumor cells and inducing suppression of antitumor immunity. Signal regulatory protein alpha should be considered as a potential therapeutic target in ESCC, especially if combined with PD-1-PD-L1 blockade.     

Proliferation inhibition effect of docetaxel combined with cisplatin on osteosarcoma cells

This research, studies the proliferation inhibition effect of combined use of docetaxel and cisplatin on osteosarcoma cells. The effects of docetaxel and cisplatin used alone and combined on osteosarcoma cell line HOS8603 were examined by means of cell count, morphologic observation, and flow cytometry (FCM). It was found that docetaxel and cisplatin either used alone or in combination, significantly inhibited the proliferation of osteosarcoma cells and apparently induced the apoptosis, and the effect of the combined drugs was better than that of the drugs used alone (growth inhibition ratio ≥59.34%, P < 0.05; apoptosis ratio = 18.31%, P < 0.01). Therefore, the combined use of docetaxel and cisplatin is more effective than either of the drugs used alone in inhibiting the proliferation of osteosarcoma cells.     

Targeting superoxide dismutase 1 to overcome cisplatin resistance in human ovarian cancer

Purpose  Clinical drug resistance to platinum-based chemotherapy is considered a major impediment in the treatment of human ovarian cancer. Multiple pathways associated with drug resistance have been suggested by many previous studies. Over expression of several key proteins involved in DNA repair, drug transport, redox regulation, and apoptosis has been recently reported by our group using a global quantitative proteomic profiling approach. Superoxide dismutase 1 (SOD1) is one of these proteins consistently over-expressed in cisplatin-resistant ovarian cancer cells as compared to their sensitive counterparts, but its precise role in drug resistance is yet to be defined. Method  In the current study, we examined the role of SOD1 in drug resistance by inhibiting its redox activity in cisplatin-resistant ovarian cancer cells using a small-molecule inhibitor, triethylenetetramine (TETA). The effect of TETA was determined by the cell proliferation assay, clonogenic cell survival assay, and SOD1 activity assay. Results  The inhibition of the SOD1 activity enhanced the cisplatin sensitivity in the resistant cells supporting the hypothesis that SOD1 is a key determinant of cisplatin resistance and is an exploitable target to overcome cisplatin drug resistance. Conclusion  SOD1 plays an important role in cisplatin resistance and modulation of its activity may overcome this resistance and ultimately lead to improved clinical outcomes.     

Podoplanin is expressed at the invasive front of esophageal squamous cell carcinomas and is involved in collective cell invasion

The expression of podoplanin is reportedly involved in collective cell invasion, which is independent from the epithelial–mesenchymal transition (EMT). We focused on the expression of podoplanin in esophageal squamous cell carcinomas (ESCC) and investigated the correlation of podoplanin and EMT‐related markers, and evaluated its prognostic significance. Five ESCC cell lines were subjected to western blot analysis for podoplanin and EMT markers. The effects of podoplanin on EMT and carcinoma invasion were evaluated with wound healing assays, invasion assays and 3‐D culture. Transfection of ectopic podoplanin into a podoplanin‐negative ESCC cell line (TE‐15) induced cell migration and invasive activity (P < 0.001 and P < 0.05, respectively) without downregulation of E‐cadherin. In contrast, transfection of si‐podoplanin RNA into a podoplanin‐positive ESCC cell line (TE‐13) reduced cell migration and invasive activity (P < 0.05). We reviewed 101 patients who had undergone esophagectomy for ESCC. Podoplanin expression was observed in 58 patients (57.4%), and positive expression was positively correlated with expression of E‐cadherin (P < 0.01), deeper wall invasion (P < 0.01), venous invasion (P < 0.05) and poorer prognosis (P < 0.01). Multivariate Cox analysis revealed that expression of podoplanin was a significant and independent unfavorable predictor of survival (P < 0.05). These data suggest that podoplanin is significantly associated with and likely contributes to ESCC invasion in the absence of EMT.     

Uptake of positron emission tomography tracers reflects the tumor immune status in esophageal squamous cell carcinoma

The relationship between the local immune status and cancer metabolism regarding ¹⁸F‐FDG and ¹⁸F‐FAMT uptake in esophageal squamous cell carcinoma (ESCC) remains unknown. The present study examined the correlations between tumor immune status, clinicopathological factors, and positron emission tomography (PET) tracer uptake in ESCC. Forty‐one ESCC patients who underwent ¹⁸F‐FDG PET and ¹⁸F‐FAMT PET before surgery were enrolled in the study. Immunohistochemistry was conducted for programmed death 1 (PD‐1), CD8, Ki‐67, CD34, GLUT1 (¹⁸F‐FDG transporter) and LAT1 (¹⁸F‐FAMT transporter). ESCC specimens with high tumoral PD‐L1 and high CD8‐positive lymphocytes were considered to have “hot tumor immune status.” High PD‐L1 expression (53.7%) was significantly associated with tumor/lymphatic/venous invasion (P = 0.028, 0.032 and 0.018), stage (P = 0.041), CD8‐positive lymphocytes (P < 0.001), GLUT1 (P < 0.001), LAT1 expression (P = 0.006), Ki‐67 labelling index (P = 0.009) and CD34‐positive vessel counts (P < 0.001). SUVmax of ¹⁸F‐FDG was significantly higher in high PD‐L1 cases than in low PD‐L1 cases (P = 0.009). SUVmax of ¹⁸F‐FAMT was significantly higher in high PD‐L1 (P < 0.001), high CD8 (P = 0.012) and hot tumor groups (P = 0.028) than in other groups. High SUVmax of ¹⁸F‐FAMT (≥4.15) was identified as the only predictor of hot tumor immune status. High PET tracer uptake was significantly associated with cancer aggressiveness and hot tumor immune status in ESCC. PET imaging may be an effective tool to predict tumor immune status in ESCC with respect to immune checkpoint inhibitor sensitivity.     

Association of GSTP1 CpG Islands Hypermethylation with Poor Prognosis in Human Breast Cancers

Glutathione S-transferase P1 (GSTP1) belongs to a family of phase II metabolic enzymes that can detoxify the carcinogens and cytotoxic drugs by conjugating them with glutathione. Relationship of GSTP1 CpG islands hypermethylation or GSTP1 protein expression with clinicopathological characteristics of breast cancers was studied. The CpG islands hypermethylation status of GSTP1 gene was studied by methylation specific primer assay and GSTP1 expression was studied by immunohistochemistry in primary breast cancers. Of 174 breast tumors, 24 (14%) were found to have GSTP1 CpG islands hypermethylation. A significant association was found between GSTP1 CpG islands hypermethylation and large tumor size (P < 0.01) or lymph node metastases (P < 0.05). Relapse-free survival (RFS) rates of patients with GSTP1 CpG islands hypermethylation were significantly poorer than those without it (5-year RFS rates; 60 vs. 86%, P < 0.01). Multivariate analysis showed that GSTP1 CpG islands hypermethylation status was a statistically significant prognostic factor being independent of other conventional prognostic factors. Immunohistochemical study showed that here was a significant association between GSTP1 CpG islands hypermethylation and loss of GSTP1 expression (P < 0.01) but that RFS rates of patients with GSTP1 positive tumors were not significantly different from those with GSTP1 negative tumors. GSTP1 CpG islands hypermethylation, but not GSTP1 protein expression, is associated with a poor prognosis of breast cancers, suggesting that GSTP1 CpG islands hypermethylation has a potential to serve as a clinically useful prognostic factor.     

BNC1调控食管鳞状细胞癌细胞的恶性生物学行为及其可能的作用机制

目的：探讨碱性核蛋白1(BNC1)对食管鳞状细胞癌（ESCC）细胞增殖、迁移、侵袭、细胞周期和凋亡的影响及其作用机制。方法：通过q PCR法检测ESCC细胞和正常食管上皮细胞中BNC1 m RNA的表达水平,免疫组织化学染色法检测10例ESCC患者癌及癌旁组织中BNC1的蛋白表达水平。利用si RNA敲低BNC1在KYSE-150和KYSE-30细胞中的表达,CCK-8法、划痕愈合实验、Transwell实验和流式细胞术检测BNC1对细胞增殖、迁移、侵袭、细胞周期和凋亡等的影响。通过CHIP-seq实验和GEPIA在线网站数据分析并结合敲低BNC1后的转录组测序数据分析筛选BNC1调控的下游靶基因,q PCR法验证BNC1敲低后靶基因的表达变化,并用双荧光素酶报告基因实验验证BNC1对靶基因的调控作用。结果：BNC1 m RNA和蛋白水平在ESCC组织中较癌旁组织高表达（均P<0.01）。敲低BNC1可明显抑制KYSE-150、KYSE-30细胞的增殖、迁移和侵袭能力（P<0.05或P<0.01）,将细胞阻滞于G1期并促进细胞的凋亡（均P<0.01）。CHIP-...     

Prognostic value of β-catenin,c-myc,and cyclin D1 expressions in patients with esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most frequently diagnosed malignant tumors in North China. We have identified that Wnt2/β-catenin pathway is activated in ESCC cells and sodium nitroprusside (SNP) and siRNA against β-catenin not only inhibit the expressions of β-catenin and its major downstream effectors including c-myc and cyclin D1 but induce cell cycle arrest and apoptosis. The purpose of the present study was to analyze the relationship between pathological parameters including invasion depth and lymph node metastasis and the expressions of β-catenin, c-myc, and cyclin D1 in order to evaluate their values of prognosis in patients with ESCC. The expressions of β-catenin, c-myc, and cyclin D1 were detected immunohistochemically in the resected cancer tissues from 40 patients with ESCC. The β-catenin expression was reduced in 22 (55.0%) patients, which was closely correlated with invasion depth (P = 0.023) and lymph node metastasis (P = 0.003). There was the positive c-myc expression in 21 (52.5%), which was significantly correlated with invasion depth (P = 0.009) and lymph node metastasis (P = 0.001). Furthermore, the results of survival rates analyzed by Kaplan–Meier curve revealed that patients with the reduced expression of β-catenin had a poorer prognosis than those with the preserved expression (P = 0.031), and patients with the positive expression of c-myc also had a significantly poorer prognosis than those with the negative expression (P = 0.008). These findings demonstrate that β-catenin pathway plays a crucial role in the progression of ESCC, suggesting that both β-catenin and c-myc may be used as markers for predicting the prognosis of patients with ESCC.     

Glutathione S-transferase P1 (GSTP1) directly influences platinum drug chemosensitivity in ovarian tumour cell lines

Background:

Chemotherapy response in ovarian cancer patients is frequently compromised by drug resistance, possibly due to altered drug metabolism. Platinum drugs are metabolised by glutathione S-transferase P1 (GSTP1), which is abundantly, but variably expressed in ovarian tumours. We have created novel ovarian tumour cell line models to investigate the extent to which differential GSTP1 expression influences chemosensitivity.

Methods:

Glutathione S-transferase P1 was stably deleted in A2780 and expression significantly reduced in cisplatin-resistant A2780DPP cells using Mission shRNA constructs, and MTT assays used to compare chemosensitivity to chemotherapy drugs used to treat ovarian cancer. Differentially expressed genes in GSTP1 knockdown cells were identified by Illumina HT-12 expression arrays and qRT–PCR analysis, and altered pathways predicted by MetaCore (GeneGo) analysis. Cell cycle changes were assessed by FACS analysis of PI-labelled cells and invasion and migration compared in quantitative Boyden chamber-based assays.

Results:

Glutathione S-transferase P1 knockdown selectively influenced cisplatin and carboplatin chemosensitivity (2.3- and 4.83-fold change in IC₅₀, respectively). Cell cycle progression was unaffected, but cell invasion and migration was significantly reduced. We identified several novel GSTP1 target genes and candidate platinum chemotherapy response biomarkers.

Conclusions:

Glutathione S-transferase P1 has an important role in cisplatin and carboplatin metabolism in ovarian cancer cells. Inter-tumour differences in GSTP1 expression may therefore influence response to platinum-based chemotherapy in ovarian cancer patients.     

High cofilin-1 levels correlate with cisplatin resistance in lung adenocarcinomas

High cofilin-1 levels have been shown to be an accurate prognostic biomarker in non-small cell lung cancer (NSCLC) and a predictive factor in drug resistance. Herein we explore the role of cofilin-1 in cis-diamminedichloroplatinum(II) (cisplatin) resistance. We evaluated cofilin-1 levels in intrinsically cisplatin-resistant A549 (ICR-A549) cells and determined the cisplatin toxicity in A549 cells transiently transfected and overexpressing CFL1 plasmid. Moreover, expression levels (activity) of the CFL1 gene network were analyzed in a cisplatin-resistant human lung adenocarcinoma cell panel. ICR-A549 cells, selected by challenging parental cells with 10-fold drug GI₅₀ value, presented a sixfold increase in cisplatin GI₅₀ value and an increased cofilin-1 immunocontent (P?<?0.01). In addition, cells transfected with cofilin-1 became more resistant to cisplatin (P?<?0.01). High activity of the CFL1 gene network was found in a cisplatin-resistant adenocarcinoma cell panel (P?<?0.01). In vitro evidences suggest that cofilin-1 is a biological predictor of cisplatin resistance, supporting new treatment initiatives based on cofilin-1 levels to guide chemotherapeutic interventions in NSCLC patients.     

The Expression of RECK mRNA and Protein in Esophageal Squamous Cell Carcinoma and Its Clinical Significance

目的:探讨食管鳞癌组织中RECK mRNA和蛋白的表达情况及其与临床病理因素的关系。方法:应用RT-PCR和免疫组化SP法检测62例食管鳞癌组织、31例癌旁不典型增生组织及62例正常食管粘膜组织中mRNA和蛋白的表达。结果:在食管鳞癌癌变过程中RECK在癌组织、癌旁不典型增生组织及正常粘膜组织中mRNA的含量依次增高,分别为1.052±0.078、1.274±0.235、1.306±0.121,组间比较有明显差异(F=49.936,P<0.05);不同分化程度、不同浸润深度及有无淋巴结转移的食管鳞癌组织之间RECK mRNA相对含量差异均有统计学意义(F=5.081,F=26.084,U=24.011,P均<0.05)。食管鳞癌组织及癌旁不典型增生组织中RECK蛋白表达均低于正常粘膜组织,表达量分别为59.7%(37/62)、71.0%(22/31)、85.5%(53/62),组间比较有明显差异(χ2=10.331,P<0.01);食管鳞癌组织中RECK蛋白表达与癌组织的分化程度、不同浸润深度及有无淋巴结转移密切相关(P<0.05)。结论:食管鳞癌组织中RECK mRNA和蛋白表达均降低,其低表达可能与食管鳞癌发生有关。检测RECK mRNA及蛋白的表达可望成为食管鳞癌早期诊断和判断预后的分子指标之一。     

Small proline-rich repeat protein 3 enhances the sensitivity of esophageal cancer cells in response to DNA damage-induced apoptosis

Small proline-rich repeat protein 3 (SPRR3) has been linked with the altered chemoradiosensitivity, however the underlying molecular mechanisms remain elusive. Here, we report that ectopic overexpression of SPRR3 enhanced the sensitivity of cells in response to DNA damage-induced apoptosis via loss of mitochondrial membrane potential (MMP), and increasing activation of caspase 3 in human esophageal cancer cell lines. Conversely, siRNA knockdown of SPRR3 reduced apoptosis. We found that SPRR3 was localized in mitochondria and interacted with Bcl-2 in vivo, thus facilitating Bax mitochondrial translocation and the subsequent release of cytochrome c, and thereby enhancing cell sensitivity to DNA damage stimuli. In clinical samples, expression of SPRR3 was associated with the pathologic response (P = 0.007 in radiotherapy group, P = 0.035 in preoperative radiotherapy group) and good survival of patients with locally advanced esophageal squamous cell carcinoma (ESCC, P = 0.008). Taken together, our results implicate that SPRR3 might serve as a radiation-sensitive predictor of ESCC.     

Association of β-catenin,Wnt1, Smad4, Hoxa9, and Bmi-1 with the prognosis of esophageal squamous cell carcinoma

In our previous study, Human Signal Transduction in Cancer Gene Array was used in 12 fresh tumor samples to detect the gene expression profiles in the esophageal squamous cell carcinoma (ESCC) tissues matched adjacent non-cancerous samples. Among genes up-regulated at least twofold, β-catenin, Wnt1, Smad4, Hoxa9, and Bmi-1 were found. So subsequently, the aim of this study was to investigate the prognosis and clinicopathologic roles of β-catenin, Wnt1, Smad4, Hoxa9, and Bmi-1 in ESCC tissue. The mRNA and protein expression levels of β-catenin, Wnt1, Smad4, Hoxa9, and Bmi-1 genes in 70 ESCC and adjacent non-cancerous paraffin-embedded samples were determined by Real-Time Quantitative PCR (RT-PCR) and immunohistochemical staining. The mRNA expression level of β-catenin, Wnt1, Smad4, Hoxa9, and Bmi-1 in ESCC was significantly higher than that in the adjacent non-cancerous tissues (0.0821 ± 0.0416 vs. 0.0185 ± 0.0201, P = 0.0000; 1.9934 ± 1.9888 vs. 0.8863 ± 0.665, P = 0.0184; 0.0298 ± 0.0215 vs. 0.0189 ± 0.0187, P = 0.0017; 2.098 ± 0.091 vs. 1.016 ± 0.078, P = 0.0000; 2.181 ± 2.158 vs. 0.931 ± 0.894, P = 0.0152; respectively), and the protein expression level of determined genes was also significantly higher than that in the adjacent non-cancerous tissues (0.2835 ± 0.0844 vs. 0.2352 ± 0.0670, P = 0.0003; 0.3830 ± 0.0947 vs. 0.2721 ± 0.1474, P = 0.0000; 0.2637 ± 0.0348 vs. 0.2042 ± 0.0180, P = 0.0000; 0.2058 ± 0.0316 vs. 0.1218 ± 0.0518, P = 0.0000; 0.2736 ± 0.0834 vs. 0.2251 ± 0.0571, P = 0.0001; respectively). Then, the overexpression of mRNA and protein levels of β-catenin, Wnt1 and Bmi-1 was aggressively associated with lymph node metastasis, advanced pathological stage, and prognosis of the patients with ESCC (P < 0.05). The up-expression of Hoxa9 mRNA and protein was also aggressively associated with lymph node metastasis and advanced pathological stage (P < 0.05); however, the overexpression of Hoxa9 protein was not associated with the prognosis (P > 0.05). Meanwhile, the hypo-expression of Smad4 mRNA was aggressively associated with advanced pathological stage and prognosis of the patients with ESCC (P < 0.05); however, the hypo-expression of Smad4 protein was neutral to the prognosis and lymph node metastasis (P > 0.05). β-catenin, Wnt1, Smad4, Hoxa9, and Bmi-1 protein expression analysis showed that the positive outcomes of the combined detection of Wnt1 and β-catenin expression or Wnt1, β-catenin and Bmi-1 expression were significantly worse than those of a single target protein expression (P < 0.05). Meantime, the prognosis of the combined positive expression of Wnt1, β-catenin, and Bmi-1 was poorer than that in the combined positive expression of Wnt1 and β-catenin (P < 0.05). The prognosis of ESCC patients with the overexpression of Wnt1/β-catenin and Bmi-1 was relatively poor, and the level of Wnt1/β-catenin and Bmi-1 was conversely correlated with advanced pathological stage and lymph node metastasis. The expression level of Smad4 and Hoxa9 mRNA was also associated with the prognosis of the patients with ESCC, pathological stage, and lymph node metastasis; however, they might not be the independent prognostic factor.