Potential Therapeutic Target For Esophageal Squamous Cell Carcinoma Research Articles

MMP1 promotes tumor growth and metastasis in esophageal squamous cell carcinoma

Matrix metalloproteinases play an essential role in the progression of esophageal squamous cell carcinoma (ESCC). Here, we show that MMP1 expression was markedly increased in a majority of ESCC compared with nontumorous tissue. High expressions of MMP1 were closely associated with lymph node metastasis, microvessel density and advanced TNM stage. Kaplan–Meier and multivariate analyses indicated MMP1 as an independent factor for overall survival in two independent cohorts of 613 patients with ESCC. In vitro studies demonstrated that MMP1 overexpression resulted in enhanced cell viability, abilities of colony formation and cell migration. The knockdown of MMP1 in ESCC cells resulted in the opposite phenomenon. Consistently, in vivo data showed that ectopic expression of MMP1 promoted tumor growth and metastasis. Further study revealed that MMP1 facilitated ESCC through the activation of the PI3K/AKT pathway. Inhibition of the PI3K/AKT pathway by LY294002 significantly attenuated MMP1-mediated cell proliferation and migration. Taken together, our data suggest that MMP1 functions as an oncogene and serves as a prognostic biomarker and a potential therapeutic target in ESCC.

MicroRNA-186 inhibits cell proliferation and induces apoptosis in human esophageal squamous cell carcinoma by targeting SKP2

miR-186 has been demonstrated to have a significant role as a tumor suppressor in many types of cancers. Nevertheless, its biological function in esophageal squamous cell carcinoma (ESCC) remains unknown. In the present study, we found that the expression level of miR-186 was downregulated in ESCC in comparison with the adjacent normal tissues and was significantly associated with differentiation level, TNM stage, and lymph node metastasis of ESCC. Functional experiments revealed that enforced overexpression of miR-186 in ESCC cells suppressed the proliferation, invasion, and induced the apoptosis of cells. Luciferase reporter assay and western blotting analysis were performed to verify the target gene regulated by miR-186, SKP2. Our findings established that the miR-186 has a suppressive role in ESCC progression via SKP2-mediated pathway, and this implies that miR-186 could be a potential therapeutic target for ESCC.

Decreased Expression of FADS1 Predicts a Poor Prognosis in Patients with Esophageal Squamous Cell Carcinoma.

FADS1 (fatty acid desaturase 1) plays a crucial role in fatty acid metabolism, and it was recently reported to be involved in tumorigenesis. However, the role of FADS1 expression in esophageal squamous cell carcinoma (ESCC) remains unknown. In the current study, we investigated the expression and clinical pathologic and prognostic significance of FADS1 in ESCC. Immunohistochemical analyses revealed that 58.2% (146/251) of the ESCC tissues had low levels of FADS1 expression, whereas 41.8% (105/251) exhibited high levels of FADS1 expression. In positive cases, FADS1 expression was detected in the cytoplasm of cells. Correlation analyses demonstrated that FADS1 expression was significantly correlated with tumor location (p=0.025) but not with age, gender, histological grade, tumor status, nodal status or TNM staging. Furthermore, patients with tumors expressing high levels of FADS1had a longer disease-free survival time (p<0.001) and overall survival time (p<0.001). Univariate and multivariate analyses revealed that, along with nodal status, FADS1 expression was an independent and significant predictive factor (p<0.001). In conclusion, our study suggested that FADS1 might be a valuable biomarker and potential therapeutic target for ESCC.

Phosphotyrosine profiling identifies ephrin receptor A2 as a potential therapeutic target in esophageal squamous-cell carcinoma.

Esophageal squamous‐cell carcinoma (ESCC) is one of the most common malignancies in Asia. Currently, surgical resection of early‐stage tumor is the best available treatment. However, most patients present late when surgery is not an option. Data suggest that chemotherapy regimens are inadequate for clinical management of advanced cancer. Targeted therapy has emerged as one of the most promising approaches to treat several malignancies. A prerequisite for developing targeted therapy is prior knowledge of proteins and pathways that drive proliferation in malignancies. We carried out phosphotyrosine profiling across four different ESCC cell lines and compared it to non‐neoplastic Het‐1A cell line to identify activated tyrosine kinase signaling pathways in ESCC. A total of 278 unique phosphopeptides were identified across these cell lines. This included several tyrosine kinases and their substrates that were hyperphosphorylated in ESCC. Ephrin receptor A2 (EPHA2), a receptor tyrosine kinase, was hyperphosphorylated in all the ESCC cell lines used in the study. EPHA2 is reported to be oncogenic in several cancers and is also known to promote metastasis. Immunohistochemistry‐based studies have revealed EPHA2 is overexpressed in nearly 50% of ESCC. We demonstrated EPHA2 as a potential therapeutic target in ESCC by carrying out siRNA‐based knockdown studies. Knockdown of EPHA2 in ESCC cell line TE8 resulted in significant decrease in cell proliferation and invasion, suggesting it is a promising therapeutic target in ESCC that warrants further evaluation.

Effect and mechanism of RUNX3 gene on biological characteristics of human esophageal squamous cell carcinoma (ESCC).

The aim of this study was to investigate the role of RUNX3 in esophageal squamous cell carcinoma (ESCC) cells biological behavior and the relationship between the expression of RUNX3 and MMP-9, TIMP-1, ICAM-1. RUNX3 levels in 90 esophageal squamous cell carcinoma specimens using immunohistochemical staining to examine the correlation between RUNX3 expression and clinical stage of ESCC. Furthermore, the role of RUNX3 in ESCC progression was evaluated in vitro by siRNA-mediated knockdown of RUNX3 or lentivirus-mediated over-expression of RUNX3 in ESCC cell lines. The expression and activities of MMP-9, TIMP-1, and ICAM-1 were analyzed. We found decreased expression of RUNX3 in ESCC tissue to be significantly related to T stage of tumor (p<0.01). In vitro, knockdown of RUNX3 in Eca9706 cells resulted in promoting cell growth, migration, and invasion. Additionally, MMP-9 and ICAM-1 were upregulated in RUNX3-knockdown cells. Notably, RUNX3 over-expression in Kyse150 cells could significantly decrease MMP-9 and ICAM-1. Tumorigenesis in vivo was significantly determined. The study indicates that low expression of RUNX3 in human ESCC tissue is significantly correlated with progression. Restoration of RUNX3 expression significantly inhibits ESCC cells migration, invasion, and tumorigenesis, which may be caused by RUNX3's interaction with MMP-9 and ICAM-1; RUNX3 may be a potential therapeutic target for ESCC.

MicroRNA-9 promotes tumor metastasis via repressing E-cadherin in esophageal squamous cell carcinoma.

MicroRNAs (miRNAs) play a critical role in development and progression of cancers. Deregulation of MicroRNA-9 (miR-9) has been documented in many types of cancers but their role in the development of esophageal squamous cell carcinoma (ESCC) has not been studied. This study aimed to investigate the effect of miR-9 in esophageal cancer metastasis. The up-regulation of miR-9 was frequently detected in primary ESCC tumor tissue, which was significantly associated with clinical progression (P = 0.022), lymph node metastasis (P = 0.007) and poor overall survival (P < 0.001). Functional study demonstrated that miR-9 promoted cell migration and tumor metastasis, which were effectively inhibited when expression of miR-9 was silenced. Moreover, we demonstrated that miR-9 interacted with the 3'-untranslated region of E-cadherin and down-regulated its expression, which induced β-catenin nuclear translocation and subsequently up-regulated c-myc and CD44 expression. In addition, miR-9 induced epithelial-mesenchymal transition (EMT) in ESCC, a key event in tumor metastasis. Taken together, our study demonstrates that miR-9 plays an important role in ESCC metastasis by activating β-catenin pathway and inducing EMT via targeting E-cadherin. Our study also suggests miR-9 can be served as a new independent prognostic marker and/or as a novel potential therapeutic target for ESCC.

Low SOX17 expression is a prognostic factor and drives transcriptional dysregulation and esophageal cancer progression

The transcriptional network of the SRY (sex determining region Y)-box 17 (SOX17) and the prognostic impact of SOX17 protein expression in human cancers remain largely unclear. In this study, we evaluated the prognostic effect of low SOX17 protein expression and its dysregulation of transcriptional network in esophageal squamous cell carcinoma (ESCC). Low SOX17 protein expression was found in 47.4% (73 of 154) of ESCC patients with predicted poor prognosis. Re-expression of SOX17 in ESCC cells caused reduced foci formation, cell motility, decreased ESCC xenograft growth and metastasis in animals. Knockdown of SOX17 increased foci formation in ESCC and normal esophageal cells. Notably, 489 significantly differential genes involved in cell growth and motility controls were identified by expression array upon SOX17 overexpression and 47 genes contained putative SRY element in their promoters. Using quantitative chromatin immunoprecipitation-PCR and promoter activity assays, we confirmed that MACC1, MALAT1, NBN, NFAT5, CSNK1A1, FN1 and SERBP1 genes were suppressed by SOX17 via the SRY binding-mediated transcriptional regulation. Overexpression of FN1 and MACC1 abolished SOX17-mediated migration and invasion suppression. The inverse correlation between SOX17 and FN1 protein expression in ESCC clinical samples further strengthened our conclusion that FN1 is a transcriptional repression target gene of SOX17. This study provides compelling clinical evidence that low SOX17 protein expression is a prognostic biomarker and novel cell and animal data of SOX17-mediated suppression of ESCC metastasis. We establish the first transcriptional network and identify new suppressive downstream genes of SOX17 which can be potential therapeutic targets for ESCC.

The role of NF-κB pathway in cancer inflammation of esophageal squamous carcinoma.

42 Background: Chronic inflammation plays an important role in tumorigenesis and tumor progression in human cancers. We aim to investigate the role of NF-kB in cancer inflammation of esophageal squamous cell carcinoma (ESCC). Methods: To generate M2-polarized macrophages, cells of human U937 monocyte cell line were treated with phorbol myristate acetate (PMA, 50 ng/ml) for 6 hours, and then cultured with PMA plus Th2 cytokines, IL-4 (20 ng/ml) and IL-13 (20 ng/ml), for another 18 hours. M2 phenotype was verified by flow cytometry and by cytokine profiling using enzyme-linked immunosorbent assay (ELISA). After co-culture with M2 macrophages, transcription nuclear factor-kB (NF-kB) activity was measured using quantitative polymerase chain reaction (Q-PCR), followed by reconfirmation with Western blot analysis for IkBα in KYSE-170 and -510 ESCC cell lines (kindly provided by Dr. Yutaka Shimada at Kyoto University, Japan). A selective inhibitor to NF-kB, Bay11-7082, was used to treat ESCC cell lines co-cultured with M2 macrophages, followed by cell proliferation, migration, invasion assays and vascular endothelial growth factor (VEGF) secretion by ELISA. The effect of Bay11-7082 (5 mg/kg) against growth of ESCC tumor was tested in xenografted tumors. Results: PMA plus Th2 cytokines treatment promoted differentiation of U937 cells into M2 macrophages. When treated with Bay11-7082, proliferation, migration, invasion and induction of VEGF expression was significantly inhibited in M2 macrophage co-cultured ESCC cells with a down-regulation of IkBα expression. Tumor growth was significantly increased in M2 macrophage co-cultured ESCC cells compared to that of the non-co-cultured controls, which was significantly retarded by treatment with Bay11-7082. Conclusions: NF-kB pathway was activated in ESCC cell lines co-cultured with M2 macrophages with an increase in cell proliferation, cell motility and angiogenic factor in vitro and tumor growth in vivo, which were significantly suppressed by a NF-kB inhibitor, Bay11-7082. These results suggest a role of M2 macrophage in promoting aggressiveness of ESCC cells, possibly through an activation of NF-kB pathway that may serve as a potential therapeutic target for ESCC.

Amplification of the telomerase RNA component gene as a new genetic marker for disease progression and prognosis in esophageal squamous cell carcinoma

Amplification of the human telomerase RNA component (TERC) gene was found in esophageal squamous cell carcinoma (ESCC). However, its roles in the progression and prognosis of ESCC have not been well understood. The amplification of TERC in normal mucosa, low-grade and high-grade intraepithelial neoplasia, and invasive ESCC samples were evaluated using a fluorescence in situ hybridization assay. The amplification of TERC invariably occurred in high-grade intraepithelial neoplasia and invasive ESCC, partially occurred in low-grade intraepithelial neoplasia specimens, and seldom occurred in normal mucosa. The average signal ratio of TERC to chromosome 3 centromere-specific probe (TERC/CSP3) was 1.00 ± 0.01 (average ± standard deviation) in normal mucosas, 1.01 ± 0.08 in low-grade intraepithelial neoplasias, 1.39 ± 0.26 in high-grade intraepithelial neoplasias, and 1.56 ± 0.41 in invasive ESCC. High TERC/CSP3 ratio was positively associated with lymph node metastasis (P = 0.005) and advanced tumor stage (P = 0.045). Patients with high amplification of TERC had poor survival (P = 0.01). The amplification of TERC could be used as a new genomic marker for disease progression and prognosis of ESCC. The amplified TERC gene may be a potential therapeutic target for ESCC.

LDHA is necessary for the tumorigenicity of esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most common lethal tumors in the world, and the development of new therapeutic targets is needed. Recent studies have shown that aerobic glycolysis, also known as the Warburg effect, mediated the anti-apoptotic effects in cancer cells. Lactate dehydrogenase A (LDHA) which executed the final step of aerobic lactate production has been reported to be involved in the tumor progression. However, the function of LDHA in ESCC has not been investigated. In this study, it was found that LDHA was up-regulated in ESCC clinical samples. Knockdown of the expression of LDHA inhibited cell growth and cell migration in vitro as well as tumorigenesis in vivo. With regard to the molecular mechanism, silencing the expression of LDHA was related to decreased AKT activation and cyclin D1 expression and increased cleavage of PARP and caspase 8. Taken together, our findings suggest that LDHA plays an important role in the progression of ESCC by modulating cell growth, and LDHA might be a potential therapeutic target in ESCC.

Overexpression of SMYD2 relates to tumor cell proliferation and malignant outcome of esophageal squamous cell carcinoma

Although we have identified two putative targets, ATF3 and CENPF, for a frequently gained/amplified region around 1q32-q41 in esophageal squamous cell carcinoma (ESCC), it is possible that other amplification targets remain to be identified. In this study, we tested whether SET and MYND domain-containing protein 2 (SMYD2), located between those two genes and encoding a lysine methyltransferase for histone H3K36 and p53K370 that regulates transcription and inhibits transactivation activity, respectively, acts as a cancer-promoting gene through activation/overexpression in ESCC. Frequent overexpression of SMYD2 messenger RNA and protein was observed in KYSE150 cells with remarkable amplification at 1q32-41.1 and other ESCC cell lines (11/43 lines, 25.6%). Overexpression of SMYD2 protein was frequently detected in primary tumor samples of ESCC (117/153 cases, 76.5%) as well and significantly correlated with gender, venous invasion, the pT category in the tumor-lymph node-metastases classification and status of recurrence. Patients with SMYD2-overexpressing tumors had a worse overall rate of survival than those with non-expressing tumors, and SMYD2 positivity was independently associated with a worse outcome in the multivariate analysis. Knockdown of SMYD2 expression inhibited and ectopic overexpression of SMYD2 promoted the proliferation of ESCC cells in a TP53 mutation-independent but SMYD2 expression-dependent manner. These findings suggest that SMYD2 plays an important role in tumor cell proliferation through its activation/overexpression and highlight its usefulness as a prognosticator and potential therapeutic target in ESCC.

Overexpression of PLK1 is associated with poor survival by inhibiting apoptosis via enhancement of survivin level in esophageal squamous cell carcinoma

PLK1 is essential for the maintenance of genomic stability during mitosis. In our study, we found that overexpression of PLK1 was an independent prognostic factor (RR=4.253, p=0.020) and significantly correlated with survivin, an antiapoptotic protein, in esophageal squamous cell carcinoma (ESCC). Reverse transcription-polymerase chain reaction and fluorescence in situ hybridization (FISH) revealed upregulation of PLK1 mRNA and amplification of PLK1 gene, respectively. Depletion of PLK1 activated the intrinsic apoptotic pathway, which was substantiated by loss of mitochondrial membrane potential, reduction of Mcl-1 and Bcl-2 as well as activation of caspase-9. Coimmunoprecipitation and confocal microscopy displayed that PLK1 was associated with survivin and PLK1 depletion led to downregulation of survivin. Cotransfection of survivin constructs could partially reverse PLK1-depletion-induced apoptosis. These data suggest that PLK1 might be a useful prognostic marker and a potential therapeutic target for ESCC. Survivin is probably involved in antiapoptotic function of PLK1.

The Suppression of Aurora-A/STK15/BTAK Expression Enhances Chemosensitivity to Docetaxel in Human Esophageal Squamous Cell Carcinoma

We previously reported that the expression of Aurora-A was frequently up-regulated in human esophageal squamous cell carcinoma (ESCC) tissues as well as cell lines and the up-regulation contributed to a poor prognosis. In this study, we assessed the possibility of Aurora-A suppression as a therapeutic target for ESCC using ESCC cell lines. We established subclones using vector-based short hairpin RNA (shRNA). Then, we investigated the effect of Aurora-A suppression on proliferation and cell cycle changes in vitro. Next, chemosensitivity against docetaxel was investigated by tetrazolium salt-based proliferation assay (WST assay) and cell number determinations, and furthermore, the type of cell death induced by docetaxel was analyzed by flow cytometry. Finally, to examine the effect of Aurora-A shRNA on proliferation and chemosensitivity against docetaxel in vivo, a s.c. tumor formation assay in nude mice was done. We established two genetically different stable cell lines (510 A and 1440 A) in which levels of Aurora-A were reduced. Cell growth was inhibited by 38.7% in 510 A and by 24.3% in 1440 A in vitro compared with empty vector-transfected controls (510 m and 1440 m), and this growth inhibition was mediated through G(2)-M arrest as confirmed by flow cytometry. Next, in a WST assay, the IC(50) for Aurora-A shRNA-transfected cells was lower than that of empty vector-transfected cells (510 A, 2.7 x 10(-7) mol/L; 510 m, 4.8 x 10(-7) mol/L; 1440 A, 2.6 x 10(-7) mol/L; 1440 m, 4.9 x 10(-7) mol/L). In addition, 0.3 nmol/L docetaxel induced a notable level of apoptosis in Aurora-A shRNA-transfected cells compared with empty vector-transfected cells. In the assay of s.c. tumors in nude mice, tumor growth in 510 A was inhibited by 36.1% compared with that in 510 m, and in tumors treated with docetaxel, the suppression of Aurora-A resulted in 44.0% tumor growth suppression in vivo. These results indicated that Aurora-A might play an important role in chemosensitivity to docetaxel, and the suppression of its expression might be a potential therapeutic target for ESCC.