Update on the Association of CD44 Expression with Esophageal Squamous Cell Carcinoma Invasion.

Role of Hyaluronan-Mediated CD44 Signaling in Head and Neck Squamous Cell Carcinoma Progression and Chemoresistance

Background: Esophageal cancer is the fourth most prevalent malignancy in China. The relationship between COX-2, CD44v6, and nm23H1 in esophageal squamous cell carcinoma (ESCC) remains unclear. Material and Methods: Expression of COX-2, CD44v6, and nm23H1 was examined, using the streptavidin-peroxidase method, in 82 ESCC and 30 normal esophageal mucosa (NEM) samples from the Shaanxi Province in China. Results: The positive rates of COX-2, CD44v6, and nm23H1 were 73.2% (60/82), 64.6% (53/82), and 24.4% (31/82), respectively in ESCC, but 6.7% (2/30), 3.3% (1/30), and 90% (27/30), respectively in NEMs. There was a statistically significant difference between NEMs and ESCCs (p < 0.05). Expression of COX-2 showed a positive statistical correlation with expression of CD44v6 (r = 0.4732, p < 0.0001), and an inverse correlation with nm23H1 (r = –0.3226, p = 0.0035). Expression of COX-2, CD44v6, and nm23H1 had no significant correlation with gender or age (p > 0.05), but increased expression of COX-2 and CD44v6 showed statistical correlation with invasion and lymph node metastasis, respectively (p < 0.05). Decreased expression of nm23H1 was statistically correlated with lymph node metastasis (p = 0.0007) but had no correlation with invasion (p = 0.8221).Conclusions: This is the first report of a significant correlation between COX-2, CD44v6, and nm23H1 in ESCC. This knowledge might help us to further understand the molecular mechanisms of carcinogenesis and progression of ESCC.

AGA Clinical Practice Update on the Utility of Endoscopic Submucosal Dissection in T1b Esophageal Cancer: Expert Review

The esophageal squamous cell carcinoma (ESCC) has high incidence in Shaanxi Province of China. More and more researches indicated that human papillomavirus type 16 (HPV16) might play an important role in carcinogenesis of ESCC but the relationship between HPV16 and CD44v6, nm23H1 has not been elucidated. HPV16 was detected by amplifying HPV16 E6 gene through polymerase chain reaction (PCR) method and the expression of CD44v6, nm23H1 in 40 ESCCs and fifteen normal esophageal mucosa (NEM) from Shaanxi Province was examined by Streptavidin-Peroxidase (SP) method using monoclonal antibody specific to CD44v6 and nm23H1. The positive rates of HPV16 E6 gene, CD44v6 and nm23H1 were 60% (24/40), 65% (26/40) and 45% (18/40) respectively in ESCCs and 26.67% (4/15), 33.33% (5/15) and 86.67% (13/15) respectively in NEMs. There exited statistical difference for HPV16, CD44v6 and nm23H1 between NEMs and ESCCs respectively (p < 0.05). The relationship between HPV16 and the expression of CD44v6 in ESCCs was statistical significance (P = 0.021), but no significant correlation was found between HPV16 and the expression of nm23H1 (P = 0.436) in ESCCs. The infection rate of HPV16 had no statistical difference in all pathological features we observed, but the expression rates of CD44v6 and nm23H1 had statistical correlation with invasion (p = 0.001, 0.013) and lymph nodes metastasis (p = 0.014, 0.002) respectively. In different histology grade of ESSCs, the relationship between HPV16 and CD44v6 was statistical significance in grade I (p = 0.044) but was not in grade II (p = 0.165) and grade III (p = 0.658), however as to the expression of nm23H1 there exited no statistical significance in all histology grades of ESCC (p > 0.05). The expression rates of CD44v6 and nm23H1 were statistically different between grade I and II (p = 0.004, 0.016) respectively and between grade I and grade III (p = 0.014, 0.020), but not statistically different between grade II and III (p = 0.792, 0.943) respectively. Our data firstly suggested that there existed the statistical relationship between the infection of HPV16 and the expression of CD44v6 in ESCCs and that HPV16 may be involved in invasion and metastasis of ESCC.

Objective To investigate the expression and clinicopathological significance of Twist1 gene in esophageal squamous cell carcinoma (ESCC), and its effect on migration and invasion of ESCC. Methods The ESCC tissues and adjacent normal tissues of 30 ESCC patients having underwent surgery were selected. The expression levels of Twist1 mRNA and protein were detected by real time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blot methods. The expression level of Twist1 mRNA in adjacent normal tissues was used as control, the relative expression levels of Twist1 mRNA in 3 ESCC cell lines K70, K140 and EC109 were detected, and the relationship between the expression level and clinicopathological features was analyzed. ESCC cell line EC109 was selected for cell scratch test and Transwell chamber invasion assay respectively. Twist1 small interfering RNA and control double stranded nonsense RNA were transfected respectively, and the changes were compared. Results The expression level of Twist1 mRNA in ESCC tissue was significantly higher than that in adjacent normal tissue (1.91 ± 0.93 vs. 0.54 ± 0.26), and there was statistical difference (P<0.01). The relative expression levels of Twist1 mRNA in 3 ESCC cell lines K70, K140 and EC109 were significantly higher than those in adjacent normal tissue (2.30 ± 0.34, 1.78 ± 0.28 and 4.37 ± 0.25 vs. 1.00 ± 0.48), and there were statistical differences (P<0.05). The expression level of Twist1 protein in ESCC tissue was significantly higher than that in adjacent normal tissue (1.35 ± 0.66 vs. 0.25 ± 0.05), and there was statistical difference (P<0.05). The relative expression level of Twist1 mRNA in ESCC tissue had correlation with lymph node metastasis and invasion degree (P<0.05). The expression levels of Twist1 mRNA in ESCC cell lines EC109 after transfected Twist1 small interfering RNA and double stranded nonsense RNA were 0.45 ± 0.16 and 1.00 ± 0.20, the express levels of Twist1 protein were 0.24 ± 0.09 and 0.58 ± 0.10, and there were statistical differences (P<0.05). The cell scratch test result showed that the migration rates of 36 and 72 h of ESCC cell line EC109 were significantly lower than those of the control double stranded nonsense RNA, after Twist1 expression was down regulated by RNA interference: (32.6 ± 4.7)% vs. (16.2 ± 6.0)% and (71.9 ± 4.7)% vs. (53.2 ± 6.6)%, there were statistical differences (P<0.05). The Transwell chamber invasion assay result showed that the total number of cells in the bottom of Twist1 through the small interfering RNA of EC109 was lower than that of the transfected control double stranded nonsense RNA (92.5 ± 8.1 vs. 32.7 ± 7.5), and there was statistical difference (P<0.05). Conclusions Twist1 gene may be highly abnormally expressed in ESCC, which has some correlation with invasion and lymph node metastasis, and may have some influences on the migration and invasion of ESCC. Key words: Esophageal Neoplasms; Carcinoma, squamous cell; Neoplasm invasiveness; Twist1 gene

An increasing number of studies have demonstrated that long non-coding (lnc)RNAs are associated with tumor invasion, metastasis and the prognosis of patients with a variety of different tumors. However, the roles of lncRNA prostate androgen regulated transcript 1 (PART1) in esophageal squamous cell carcinoma (ESCC) remain unknown. In the present study, reverse transcription-quantitative PCR was performed to investigate the levels of PART1, SRY-box transcription factor 6 (SOX6) and miR-18a-5p in ESCC tissues and cells. The functions of PART1 in ESCC were demonstrated using Cell Counting Kit-8 and Matrigel assays. Promoter activity and dual-luciferase reporter assays, RNA immunoprecipitation and western blot analyses were also used to determine the potential mechanisms of PART1 in ESCC cell lines. It was found that PART1 and SOX6 were both downregulated in ESCC tissues and cells, and their low expression levels were associated with TNM stage, lymph node metastasis and poor prognosis in patients with ESCC. Forkhead box protein P2 (FOXP2) exhibited low expression level in ESCC tissues, and its expression was positively correlated with PART1 expression level in ESCC tissues. FOXP2 was found to bind to the promoter region of PART1 to regulate its expression in ESCC cells. Functionally, PART1 overexpression suppressed cell proliferation and invasion, whereas PART1 downregulation promoted cell proliferation and invasion in the ESCC cell lines. Mechanistically, PART1 functions as a competing endogenous (ce)RNA by sponging miR-18a-5p, resulting in the upregulation of the downstream target gene, SOX6, coupled with the inactivation of the β-catenin/c-myc signaling axis, to suppress ESCC cell proliferation and invasion. In conclusion, data from the present study unveil a potential ceRNA regulatory pathway, in which PART1 affects SOX6 expression level by sponging miR-18a-5p, to ultimately suppress ESCC development and progression.

CD44 serves as a marker of cancer stem cells. Alternative splicing generates the CD44v9 isoform. Cancer stem cells are associated with the epithelial‐mesenchymal transition in cancers, although little is known about their role in esophageal squamous cell carcinoma. Here, we aimed to clarify the relationship between CD44v9 expression, the epithelial‐mesenchymal transition, and clinicopathological features of patients with esophageal squamous cell carcinoma. CD44v9 levels were higher at the tumor invasive front compared with the center of the tumor and higher in metastatic lymph nodes compared with primary tumors. High levels of CD44v9 at the tumor invasive front were significantly associated with deeper tumor invasion and shorter overall survival and recurrence‐free survival. The expression of CD44v9 was increased by treatment with transforming growth factor‐β, which induced esophageal squamous cell carcinoma cells to undergo the epithelial‐mesenchymal transition. Moreover, inhibition of CD44v9 expression decreased the migration and invasiveness of esophageal squamous cell carcinoma cells. These results indicate that the expression of CD44v9 at the tumor invasive front induced by stemness was strongly associated with the epithelial‐mesenchymal transition and poor prognosis of patients with esophageal squamous cell carcinoma. CD44v9 may therefore serve as a novel prognostic biomarker and a potential therapeutic target for esophageal squamous cell carcinoma.

EPB41L3 may play a role as a metastasis suppressor by supporting regular arrangements of actin stress fibres and alleviating the increase in cell motility associated with enhanced metastatic potential. Downregulation of epb41l3 has been observed in many cancers, but the role of this gene in esophageal squamous cell carcinoma (ESCC) remains unclear. Our study aimed to determine the effect of epb41l3 on ESCC cell migration and invasion. We investigated epb41l3 protein expression in tumour and non-tumour tissues by immunohistochemical staining. Expression in the non-neoplastic human esophageal cell line Het-1a and four ESCC cell lines - Kyse150, Kyse510, Kyse450 and Caes17 - was assessed by quantitative Polymerase Chain Reaction (qPCR) and Western blotting. Furthermore, an EPB41L3 overexpression plasmid and EPB41L3-specific small interfering RNA were used to upregulate EPB41L3 expression in Kyse150 cells and to downregulate EPB41L3 expression in Kyse450 cells, respectively. Cell migration and invasion were evaluated by wound healing and transwell assays, respectively. The expression levels of p-AKT, matrix metalloproteinase (MMP)2 and MMP9 were evaluated. Expression of epb41l3 was significantly lower in tumour tissues than in non-tumour tissues and in ESCC cell lines compared with the Het-1a cell line. Kyse450 and Caes17 cells exhibited higher expression of epb41l3 than Kyse150 and Kyse510 cells. Overexpressing epb41l3 decreased Kyse150 cell migration and invasion, whereas EPB41L3-specific small interfering RNA silencing increased these functions in Kyse450 cells. Furthermore, overexpressing epb41l3 led to downregulation of MMP2 and MMP9 in Kyse150 and Kyse510 cells. Our findings reveal that EPB41L3 suppresses tumour cell invasion and inhibits MMP2 and MMP9 expression in ESCC cells.

Objective To observe the expression of hypoxia inducible factor-1α (HIF-1α) and chemokine receptor 4 (CXCR4) in Caes-17 cells under hypoxia,and investigate the expression and clinical significance of HIF-1 α and CXCR4 in esophageal squamous cell carcinoma (ESCC) tissues and their correlation.Methods Caes-17 cells were cultured in vitro.The expression of HIF-1α and CXCR4 was detected by using fluorescence real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting under normoxia or hypoxia.The expressions of HIF-1α and CXCR4 protein was assessed by using immunohistochemistry in 106 cases of ESCC specimens with complete clinical and pathological date and 30 cases of normal esophageal mucosa tissues.The relationship between HIF-1 α or CXCR4 and pathologic features of esophageal cancer was analyzed.The correlation between HIF-1α and CXCR4 was also assessed.Results The expression of HIF-1α protein (0.86 ±0.03),CXCR4 mRNA (6.95 ±0.30),CXCR4 protein (0.56 ± 0.02) were increased under hypoxia than those under normoxia [HIF-1 α protein (0.28±0.04),CXCR4 mRNA (1.09±0.14),CXCR4 protein (0.11±0.03)] (P＜0.05).There was no significant difference in HIF-1 α mRNA (0.96 ± 0.06) between hypoxia and normoxia (1.14 ± 0.12) (P ＞ 0.05).The positive rate of HIF-1α and CXCR4 expression in ESCC tissues was obviously higher than that in normal esophageal mucosa tissues (P ＜ 0.05).The expression of HIF-1 α in ESCC tissues was associated with depth of tumor invasion,lymph node metastasis and TNM stage (P ＜ 0.05) and the expression of CXCR4 in ESCC tissues was associated with depth of tumor invasion,lymph node metastasis (P ＜ 0.05).The expression of CXCR4 was significantly correlated with the expression of HIF-1α(r =0.539,P ＜ 0.05).Conclusion Hypoxia can increase the expression level of HIF-1 α and CXCR4 in esophageal cancer cells.The CXCR4 expression has a significantly positive correlation with the HIF-1α expression in esophageal cancer.They may play synergetic roles in the genesis,development,invasion and metastasis of ESCC. Key words: Esophageal squamous carcinoma; Hypoxia; Hypoxia inducible factor-1 α; Chemokine receptor 4 ; Immunohistochemistry

The aim of the current study was to find out a clinicopathologic significance of CD44v6 over-expression in esophageal squamous cell carcinoma (ESCC), which has not been elucidated fully. Immunohistochemical expression of CD44v6 was examined for 81 ESCCs. Correlation of CD44 over-expression with the clinicopathologic features were investigated. Thirty-eight ESCCs (46.9%) had over-expression of CD44v6. The proportions of the incidence of lymph node metastasis (P=0.039), lymphatic permeation (P=0.003), and blood vessel invasion (P=0.037) in ESCCs with over-expression of CD44v6 were significantly higher than those in ESCCs without over-expression of CD44v6. The stage of the tumor in ESCCs with over-expression of CD44v6 was significantly more advanced (P=0.045). Survival rates of patients with ESCC with over-expression of CD44v6 were significantly worse (P=0.0005). Moreover, CD44v6 over-expression (P=0.048) as well as blood vessel invasion (P=0.014) and stage of the tumor ( P=0.010) were factors independently associated with the unfavorable prognosis of the patients with ESCC. Over-expression of CD44v6 can be an indicator of the malignant potential of ESCC.

Esophageal cancer is the eighth most common malignancy and sixth leading cause of cancer deaths in the world. Recent studies have shown the potential role of the aryl hydrocarbon receptor (AhR) in tumor development; however, little is known about its role in esophageal squamous cell carcinoma. In the present study, we evaluated AhR expression in esophageal tumor tissues as well as cell lines and investigated the effects of AhR activation by its agonist BNF on esophageal squamous cell carcinoma invasion using Eca109 and TE-13 cells as a model. Western blotting was performed to detect the AhR and CYP1A1 protein expression. Transwell migration assays were carried out to study the effects of BNF on esophageal squamous cell carcinoma cell invasion. AhR-specific siRNA was used to knock down the expression of AhR protein. Our results showed that AhR was highly expressed in esophageal squamous cell carcinoma tissues and cell lines when compared with its expression in normal tissue. AhR siRNA robustly decreased AhR protein expression in both Eca109 and TE-13 cells. BNF significantly inhibited invasion of human esophageal squamous cell carcinoma cells via activation of AhR. The obtained results provide critical information on the roles of BNF in mediating esophageal squamous cell carcinoma invasion. This information could be useful for future therapeutic intervention in this lethal human disease.

Background:To examine the expression level of procollagen-lysine2-oxoglutarate 5-dioxygenase 2 (PLOD2) in esophageal squamous cell carcinoma (ESCC) and analyze its correlation with clinicopathological parameters, in order to explore the mechanism of PLOD2 in regulating invasion and metastasis of ESCC.Methods:Immunohistochemistry was used to detect the expression level of PLOD2 in tumor tissues and paired adjacent tissues of 172 patients with ESCC, and the relationship between PLOD2 expression and clinicopathological parameters was analyzed. The deposition of collagen fibers in tumor was detected by Sirius red staining. The correlation between tumor stem cells and epithelial–mesenchymal transition (EMT) markers ZEB1 was analyzed by multivariate logistic regression.Results:The expression level of PLOD2 in tumor tissues of patients with ESCC (70.35%, 121/172) was significantly higher than that in paired adjacent tissues (29.65%, 51/172; P < .01). The positive expression rate of PLOD2 in ESCC was related to T classification, lymph node metastasis, and pathological tumor node metastasis of a tumor. The expression rates of ZEB1, CD44, and CD133 in ESCC were correlated with T classification, lymph node metastasis and pathological tumor node metastasis. Scarlet red staining showed that collagen fiber deposition in ESCC tissues with high expression of PLOD2 was significantly higher than that in tissues with low expression of PLOD2 (P < .01). A positive correlation was observed between the expression of PLOD2 and CD133, PLOD2 and CD44, and PLOD2 and N-cadherin (P < .01). Moreover, a negative correlation was noted between the expression of PLOD2 and E-cadherin (P < .01). The combined expression of PLOD2 and ZEB1 were independent prognostic factors for the total survival time of patients with ESCC.Conclusion:PLOD2 is highly expressed in ESCC and is closely related to tumor invasion and metastasis. The mechanism of PLOD2 for promoting invasion and metastasis of ESCC may be related to activation of the EMT signaling pathway to promote EMT and tumor stem cell transformation.

Altered expression of Sirtuin 2 (Sirt2) was associated with cancer development and progression. This study further assessed the association of Sirt2 expression with clinicopathological data and prognosis of patients with esophageal squamous cell carcinoma (ESCC) after postoperative concurrent chemoradiotherapy. Tissue specimens from 95 ESCC patients were collected for immunohistochemical analysis of Sirt2 expression, which was used to determine association with patient clinicopathological and survival data. Sirt2 protein was expressed in 53.7% of ESCC tissue specimens but only in 25.3% of normal squamous epithelium (p = 0.000). Sirt2 expression was associated with tumor invasion (p = 0.005), lymph node metastasis (p = 0.003), and advanced clinical stage (p = 0.000), but not with tumor size (p = 0.199), or differentiation (p = 0.177). Sirt2 expression was associated with poor overall and progression-free survival (p = 0.034). The multivariate analysis showed that Sirt2 expression was an independent predictor for overall survival of patients with resected ESCC followed by concurrent chemoradiotherapy (p = 0.048). Sirt2 protein expression in ESCC tissue specimens was associated with ESCC invasion, lymph node metastasis, and advanced tumor clinical stage, as well as poor overall and poor progression-free survival. Sirt2 expression is an independent prognostic predictor for ESCC patients.

Although adenocarcinoma is the most common histology type in esophageal cancer in Western countries, squamous cell carcinoma is the most common type in Eastern countries. 1The risks of esophageal squamous cell carcinoma (ESCC) are known to be old age, smoking, alcohol intake, caustic injury, and history of head and neck cancer, among others. 2ESCC is usually evenly distributed between the middle and lower esophagus and can be located as multifocal lesions. 3lthough surgical resection has been a mainstay of curative treatment modalities for ESCC, endoscopic resection (ER) has also been a curative treatment option for ESCC in indicated cases with minimal risk of lymph node metastasis (LNM).Although many studies have reported favorable clinical outcomes after ER for ESCC, additional treatment may be necessary for the cases with risk of LNM. 4,5In ESCC, the deeper the tumor invasion, the higher the risk of LNM.Although the risk of LNM in ESCC confined to epithelial layer or lamina propria is minimal, LNM is found in 10% to 40% in ESCC that invades the muscularis mucosa or submucosa. 6,7Therefore, additional treatment may be necessary for patients with incomplete ER or tumor invasion into the muscularis mucosa or deeper layers irrespective of resection margin.Surgical resection can be considered as an additional treatment option.However, it has a high morbidity and mortality rate, especially in old age.Moreover, surgical specimens often show no residual tumor or negative tumor involvement in LN dissection.If the patient has a comorbidity that can raise perioperative risk, it is difficult to consider surgical resection as an additional treatment option.Radiotherapy (RT) can be considered as another additional treatment option.RT has the effect of reducing locoregional

We immunohistochemically examined the expression of CD44 standard (CD44 st) and CD44 variant 6 (CD44 v6) in 112 cases of primary lung cancer, and their relationship to the clinical milieu, including the clinical stage. In 46 cases of squamous cell carcinoma, expression of CD44 st was observed in 45.7% of the cases, and expression of CD44 v6 was observed in 60.9%. In 43 cases of adenocarcinoma, positive staining of CD44 st and CD44 v6 was seen in 2.3% and 4.7% of the cases, respectively. None of 21 small cell carcinomas was positive for CD44 st or CD44 v6. In squamous cell carcinomas, the expression of CD44 st and CD44 v6 was observed at a rate significantly higher than in other histologic type. Most specimens positive for CD44 st stained positively for CD44 v6. Therefore, it seems likely that the CD44 expression observed in squamous cell carcinoma of the lung was a variant CD44 containing the domain encoded by variant exon 6. The expression of CD44 v6 was not related to the clinical stage. Significant association between CD44 v6 and differentiation of squamous cell carcinoma was seen; 2/7 (28.6%) for poorly differentiated, 19/31 (61.3%) for moderately differentiated, and 7/8 (87.5%) for well differentiated squamous cell carcinomas (p = 0.02 by trend test). It was previously reported that CD44 st and CD44 v6 were expressed in both normal bronchial epithelium and squamous cell metaplasia. These results suggest that the expression of CD44 v6 in squamous cell carcinoma of the lung may reflect the immunohistochemical characteristics of the tissue from which such carcinoma emerge.