Esophageal Squamous Cell Carcinoma Aggressiveness Research Articles

CircLONP2 Accelerates Esophageal Squamous Cell Carcinoma Progression via Direct MiR-27b-3p-ZEB1 Axis.

Circular RNAs (circRNAs) are important mediators in esophageal squamous cell carcinoma (ESCC) carcinogenesis. We aim to explore the functions and mechanisms of circLONP2 in ESCC progression. The circLONP2 level was evaluated in ESCC samples and cell lines. The role and mechanisms of circLONP2 in ESCC proliferation and migration were demonstrated in vitro. We found that circLONP2 was upregulated in human ESCC and predicts poor overall survival (OS) and disease-free survival (DFS). CircLONP2 promotes ESCC aggressiveness by directly interacting with miR-27b-3p, thus upregulating the expression levels of its target gene ZEB1 by suppressing miR-27b-3p activity. Therefore, we demonstrated that circLONP2/miR-27b-3p/ZEB1 axis promotes ESCC metastasis via regulating epithelial-to-mesenchymal transition (EMT)-related proteins. CircLONP2 may serve as an oncogenic circRNA and as a prognostic biomarker in ESCC progression.

Identifying a confused cell identity for esophageal squamous cell carcinoma

The cell identity of malignant cells and how they acquire it are fundamental for our understanding of cancer. Here, we report that esophageal squamous cell carcinoma (ESCC) cells display molecular features equally similar but distinct to all three types of normal esophageal epithelial cells, which we term as confused cell identity (CCI). CCI is an independent prognostic marker associated with poor prognosis in ESCC. Further, we identify tropomyosin 4 (TPM4) as a critical CCI gene that promotes the aggressiveness of ESCC in vitro and in vivo. And TPM4 creates CCI through activating the Jak/STAT-SOX2 pathway. Thus, our study suggests an unrecognized feature of ESCC cells, which might be of value for clinic prognosis and potential interference.

Correction: Downregulation of MicroRNA-644a Promotes Esophageal Squamous Cell Carcinoma Aggressiveness and Stem Cell-like Phenotype via Dysregulation of PITX2.

In the original version of this article (1), the authors found five images that were published in error: Fig. 2B, colony formation assay of KYSE-140/miR-644a; Fig. 2C, migration assay of KYSE-140/control (0h); Fig. 2D, invasion assay of KYSE-140/miR-644a; Fig. 4C, “Scramble” proportion of side population cells (top image); and Fig. 5C, alpha-tubulin Western blots (right image). The errors have been corrected in the latest online HTML and PDF versions of the article. The authors regret the errors.

MAML1 promotes ESCC aggressiveness through upregulation of EMT marker TWIST1.

Mastermind-like1 (MAML1) is the main transcriptional co-activator of Notch signaling pathway. It playsessentialroles in several pathways including MEF2C, p53, Nf-кB andWnt/β-catenin. TWIST1 is known as a regulator ofepithelial mesenchymal transition (EMT), which is considered as a primary step in promotion oftumor cell metastasis. Since concomitant expression of these genes was observed in tumors, our aim in this study was to elucidate the linkage between MAML1 and TWIST1 co-overexpression in esophageal squamous cell carcinoma (ESCC). While MAML1 silencing significantly down-regulated TWIST1, its ectopic expression up-regulated TWIST1 expression in both mRNA and protein levels in KYSE-30 cells. Expression of mesenchymal markers was increased significantly after MAML1 and TWIST1 ectopic expression, while epithelial markers expression was significantly decreased after silencing of both genes. Concomitant protein expression of MAML1 and TWIST1 was significantly observed in ESCC patients. Enforced expression of TWIST1 had no impact on MAML1 gene expression in KYSE-30 cells. The results clearly suggest transcriptional regulation of TWIST1 by MAML1 transcription factor in ESCC cells KYSE-30. Since TWIST1 is known as an EMT inducing marker, our results may revealed the mastermind behind TWIST1 function and introduced MAML1 as an upstream master regulator of TWIST1 and EMT in KYSE-30 cells.

176P - DCLK1 promotes tumour invasion and metastasis through epithelial-mesenchymal transition and the MEK/ERK pathway in esophageal squamous cell carcinoma

BackgroundDoublecortin-like kinase1 (DCLK1), a recently discovered cancer stem cell (CSC) marker in the colon and pancreatic cancer, contributes greatly to the malignancy of gastrointestinal cancers. Also, higher DCLK1 expression is reported in esophageal adenocarcinoma (EAC) compared to Barrett esophagus and normal tissue. However, studies on the effect of DCLK1 in esophageal squamous cell carcinoma (ESCC), which accounts for about 90% of diagnosed EC in Asia areas, were not yet reported. MethodsCRISPR/Cas9 genome-editing technique was used to knockout DCLK1 in ESCC cells (Kyse450, Kyse70). Subsequently, the proliferative and invasive potential of these cells was evaluated using Real-Time Cell Analyzer for proliferation, wound healing assay for migration, and transwell assay for invasion. Epithelial-mesenchymal transformation (EMT) associated transcriptional factors and MEK/ERK pathway were tested by western blot. In addition, the correlation heatmap between DCLK1 expression and EMT associated transcriptional factors, and MEK/ERK pathway was analyzed in R among 95 ESCC patients from The Cancer Genome Atla (TCGA) database. ResultsCRISPR/Cas9 technology efficiently disrupted the DCLK1 gene and abrogated its expression in ESCC cell lines. DCLK1 deficiency significantly inhibited proliferation, migration and invasion of ESCC cells. DCLK1 deletion resulted in notable molecular changes including downregulation of mesenchymal markers such as Vimentin, ZEB1, Slug and Snail, and up-regulation of epithelial marker E-cadherin. Additionally, the phosphorylation of MEK/ERK pathway was inhibited in DCLK1-KO ESCC cells. Accordingly,evidence from TCGA ESCA demonstrated that human ESCCs expressing high levels of DCLK1 correlate with activated MEK/ERK pathway signaling suggesting greater EMT transition activity. ConclusionsCRISPR/Cas9-mediated knockout of DCLK1 results in a dramatic suppression of ESCC aggressiveness via inhibiting EMT and MEK/ERK pathway. Importantly, this study demonstrates DCLK1's functional significance in ESCC for the first time and provides a theoretical basis for the development of DCLK1-based biomarkers and targeted therapies. Legal entity responsible for the studyJiannan Yao. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Abstract 62: HIC1 inhibits cells metastasis via epithelial-mesenchymal transition in esophageal squamous cell carcinoma

Abstract Invasion and metastasis are the main causes of death in patients with esophageal squamous cell carcinoma (ESCC). Epithelial-mesenchymal transition (EMT) has been reported to be closely related with tumor metastasis. Although HIC1 (hypermethylated in cancer 1) gene has been observed to be epigenetically modified in many cancers, its intrinsic role and mechanism in ESCC metastasis, especially in EMT, remains unclear. Our previous study showed that hypermethylation of the HIC1 promoter markedly elevated in metastatic ESCC tissues compared with primary and adjacent normal tissues and it was associated with poor patients’ survival. In the present study, we used immunohistochemistry (IHC) to detect the expression of HIC1 in ESCC tissues and adjacent normal tissues, and the results showed that HIC1 was down-expressed in ESCC tissues and overexpressed in adjacent normal tissues. Results from trans-well-migration and wound healing experiments showed that overexpression of HIC1 in ESCC cells (KYSE 30) can reduce cell migration and metastasis behavior. Moreover, Western blot showed that overexpression of HIC1 could down-regulate EphA2 expression, simultaneously induced the markers of EMT target changed, in which E-cadherin was observed up-regulated, Snail and Vimentin were down-regulated. In summary, our results suggests that HIC1 may regulate EphA2-SirT1-P53 signaling pathway to mitigate ESCC aggressiveness. Note: This abstract was not presented at the meeting. Citation Format: Beibei Sha, Ping Chen, Xingge Liu, Xuanyu Hu, Miaomiao Li, Pei Li. HIC1 inhibits cells metastasis via epithelial-mesenchymal transition in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 62.

TWIST1, MMP-21, and HLAG-1 co-overexpression is associated with ESCC aggressiveness.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive types of cancer, requiring reliable biomarkers for prognosis and therapeutic responsiveness. TWIST1, as an important factor responsible for metastasis of several cancers, is involved in tumor invasion and metastasis through indirectly regulation of MMP-21 expression. On the other hand, NF-ĸβ which is a regulator of HLAG-1 has direct interaction with TWIST1 protein. In this retrospective study we investigated the clinical significance of TWIST1, MMP-21, and HLAG-1 expression in ESCC, and the possible correlation between these genes and progression of the disease. The gene expression analyses of TWIST1, MMP-21, and HLA-G1 were performed by relative comparative real-time polymerase chain reaction in 58 ESCCs compared with corresponding margin-normal esophageal tissues. Significant overexpression of HLAG-1, TWIST1, and MMP-21 messenger RNA was observed in 22.4%, 41.4%, and 60.3% of tumor samples, respectively. Concomitant overexpression of TWIST1/MMP-21 and TWIST1/HLAG-1 were significantly correlated to each other in various clinicopathological features, including depth of tumor invasion, stage of tumor progression, lymphatic invasion, and grade of tumor cell differentiation ( P < 0.05). The current study is the first report of coexpression of TWIST1, MMP-21, and HLAG-1 in ESCC. Such findings suggest an oncogenic role for concomitant expression of these genes in ESCC invasion and metastasis.

Abstract 2513: FGFRL1 deficiency reduces motility and tumor growth of cells derived from esophageal squamous cell carcinoma

Abstract Fibroblast growth factor receptor-like 1 (FGFRL1) belongs to the FGFR protein family. We previously reported that FGFRL1 is highly expressed in esophageal squamous cell carcinoma (ESCC) patients with lymph node metastasis and with the depth of ESCCs. Furthermore, transient inhibition of FGFRL1 expression induces cell cycle arrest and apoptosis of ESCC cells. However, the intracellular domain of FGFRL1 lacks the tyrosine kinase domain. Thus the mechanisms of which FGFRL1 contributes to the development of ESCC are unclear. Two cell lines (Clone 15 and 21) deficient for FGFRL1 expression were established from an ESCC cell line (KYSE520) using the CRISPR-Cas9 method. We evaluated the characteristics of these two cell lines in vitro and in vivo. In these FGFRL1-deficient KYSE520 cells, actin filaments around the nucleus were observed sparsely, whereas the filaments along the plasma membranes were observed as strong as those in the parent KYSE520 cells. Depleting FGFRL1 reduced cell motility in vitro and, decelerated tumor growth in a mouse xenograft model. Microarray analysis of mRNA expression revealed that FGFRL1-depletion resulted in reduced expression of MMP-1 and FGFBP1. In conclusions, FGFRL1-deficiency reduced both cell motility and tumor growth of KYSE520 cells. This is consistent with that high expression of FGFRL1 coincident with lymph node metastasis and poor prognosis. These results demonstrate that FGFRL1 expressed in ESCC cells has a role in aggressiveness of ESCCs. Citation Format: Yoshinori Takei, Takafumi Matsumura, Kazuaki Watanabe, Hirokazu Nakamine, Tetsuo Sudo, Kazuharu Shimizu, Yutaka Shimada. FGFRL1 deficiency reduces motility and tumor growth of cells derived from esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2513.

Activation of the Dickkopf1-CKAP4 pathway is associated with poor prognosis of esophageal cancer and anti-CKAP4 antibody may be a new therapeutic drug.

Aberrant expression of the secretory protein Dickkopf1 (DKK1) is associated with poor prognosis of esophageal squamous cell carcinoma (ESCC), but the underlying mechanism of how DKK1 is involved in aggressiveness of ESCC is not clear. In this study, we show that cytoskeleton-associated protein 4 (CKAP4) functions as a DKK1 receptor in ESCC cells. Immunohistochemical analyses of ESCC revealed that both DKK1 and CKAP4 are minimally expressed in associated normal esophageal squamous mucosa of non-tumor regions, but strongly expressed in tumor lesions. Forty-six of 119 cases (38.7%) were positive for both DKK1 and CKAP4. Those expressing both proteins showed poor prognosis and relapse-free survival. Multivariate analysis demonstrated that expression of both proteins was the poor prognostic factor. The Cancer Genome Atlas data set indicated that the mRNA levels of DKK1 and CKAP4 are significantly elevated in the tumor lesions compared to non-tumor regions. DKK1 bound to CKAP4 at endogenous levels. DKK1 induced the internalization of CKAP4 from and its recycling to the plasma membrane. AKT was activated in ESCC cells in which DKK1 was highly expressed and CKAP4 was localized to the plasma membrane. Knockdown of either DKK1 or CKAP4 inhibited AKT activity and cell proliferation in vitro and xenograft tumor formation. Wild-type CKAP4 or DKK1, but not a DKK1 mutant that was unable to bind to CKAP4, rescued phenotypes induced by CKAP4 or DKK1 knockdown, respectively. The anti-CKAP4 antibody also inhibited AKT activity and suppressed xenograft tumor formation. In contrast, in ESCC cells in which DKK1 was marginally expressed, knockdown of CKAP4 or anti-CKAP4 antibody affected neither AKT activity nor cell proliferation. These findings suggest that the DKK1-CKAP4 pathway promotes ESCC cell proliferation and that CKAP4 might represent a novel therapeutic target for ESCCs expressing both DKK1 and CKAP4.

Complex Epigenetic Regulation of Chemotherapy Resistance and Biohlogy in Esophageal Squamous Cell Carcinoma via MicroRNAs.

Background: Resistance towards chemotherapy is a major obstacle in the treatment of esophageal squamous cell carcinoma (ESCC). We investigated the role of specific microRNAs in chemotherapy resistance and tumor biology. Methods: We selected three microRNAs from characteristic microRNA signatures of resistant ESCC (hsa-miR-125a-5p, hsa-miR-130a-3p, hsa-miR-1226-3p), and hsa-miR-148a-3p. Effects on chemotherapy, adhesion, migration, apoptosis and cell cycle were assessed in six ESCC cell lines. Target analyses were performed using Western blotting and luciferase techniques. Results: MiR-130a-3p sensitized cells towards cisplatin in 100% of cell lines, miR-148a-3p in 83%, miR-125a-5p in 67%, miR-1226-3p in 50% (p ≤ 0.04). MiR-130a-3p sensitized 83% of cell lines towards 5-FU, miR-148a-3p/miR-125a-5p/miR-1226-3p only 33% (p ≤ 0.015). Several resistance-relevant pathways seem to be targeted on various levels. Bcl-2 was confirmed as a direct target of miR-130a-3p and miR-148a-3p, and p53 as a target of miR-125a-5p. All microRNAs decreased migration and adhesion, except miR-130a-3p, and increased apoptosis. Simultaneous manipulation of two microRNAs exhibited additive sensitizing effects towards cisplatin in 50% (miR-125a-5p/miR-148a-3p), and 75% (miR-148a-3p/miR-130a-3p) of cell lines (p ≤ 0.006). Conclusion: Our data present strong evidence that specific microRNA signatures are responsible for drug resistance and aggressiveness of ESCC. Final functional readout of these complex processes appears to be more important than single microRNA-target interactions.

Investigation of melanoma-associated antigen A4 cancer/testis antigen clinical relevance in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is considered as the seventh most common cancer worldwide, and the second prevalent malignancy in the north of Iran. A subfamily group of tumor-specific antigens, commonly specified as cancer/testis antigens (CTAs), are expressed restrictedly in testis, ovary, and placenta. Melanoma-associated antigen A4 (MAGEA4) as a CTA is overexpressed in a variety of cancers. Expressional analysis of MAGEA4 protein in ESCC may be useful to investigate its clinical relevance leading to effective improvements in ESCC diagnosis and treatment. Fifty-six ESCC patients with no preoperative therapeutic circumstance such as radiotherapy or chemotherapy were analyzed to explore the protein expression level of MAGEA4 using immunohistochemistry assay. MAGEA4 overexpression was detected in 66% of ESCC samples showing strong nuclear and cytoplasmic staining compared to the normal epithelium. There were significant correlations between MAGEA4 protein expression and depth of tumor invasion (P = 0.019), and the number of involved lymph nodes (P = 0.045). Because of the significant correlation of MAGEA4 and indices of poor prognosis, the role of this CTA may be confirmed in ESCC aggressiveness and metastasis. Therefore, MAGEA4 may be a promising therapeutic candidate for suppressing ESCC aggressiveness.

The notch pathway is activated in neoplastic progression in esophageal squamous cell carcinoma

The Notch signaling pathway is integral to normal human development and homeostasis and has a deterministic function on cell differentiation. Recent studies suggest aberrant Notch signaling may contribute to neoplastic progression by an increase in stem cell survival, chemoresistance, and the promotion of epithelial-to-mesenchymal transition. The goals of our study were to determine, utilizing quantitative technologies, the expression of activated Notch 1 in esophageal squamous cell carcinoma (SCC) and to determine the relationship between Notch 1 expression and various clinicopathologic parameters. Immunohistochemical staining for Notch intracellular domain (NICD) was performed on 60 consecutive cases of esophageal squamous cell carcinoma, 42 cases of benign esophageal squamous epithelium, and 13 cases of eosinophilic esophagitis diagnosed in our department from 2007 through 2015, and exact nuclear staining and nuclear characteristics were graded using the Vectra imaging system. Clinicopathologic data (gender, age at diagnosis, smoking status, tumor grade, tumor stage, tumor location, and survival) were collected for each SCC case and these were correlated with NICD staining. Cases of esophageal SCC demonstrated significantly higher NICD staining compared to cases of benign and reactive esophageal epithelium (P=.003 and .005, respectively). Among cases of esophageal SCC, nuclear NICD staining was significantly correlated with both tumor grade and stage. Following classification and regression tree analysis, esophageal SCC patients with increased NICD expression were found to be more likely to die from their disease than those with lower levels of expression. Taken together, the findings suggest that increased Notch 1 may contribute to the development and aggressiveness of esophageal SCC.

Abstract 5566: African American esophageal squamous cell carcinoma expression profile reveals dysregulation of stress response and detox networks

Abstract Background: Esophageal carcinoma is the third most common gastrointestinal malignancy worldwide and is largely unresponsive to therapy. African-Americans have an increased risk for esophageal squamous cell carcinoma (ESCC), this subtype that shows a marked variation in geographic incidence. So far, the molecular architecture of African-American ESCC is still poorly understood. It is unclear why African-American ESCC is more aggressive and the survival rate of these patients worse than those of other ethnic groups. Our aim is to investigate the central question of whether there is a genetic basis for the aggressive nature of ESCC among African-Americans through analysis of genetic overlaps and differences between African-American ESCC and reported Asian data. Methods: We conducted microarray expression profiling in pairs of esophageal squamous cell tumors and matched controls to define genetic alterations that occur in African American ESCC. We then utilized Ingenuity Pathway Analysis (IPA) to analyze the differentially expressed genes in African-American ESCC. We conducted a meta-analysis of differentially expressed genes of African American ESCC and those of Asian ESCC through IPA. Results: Transcriptome profiling of African- American ESCC tumors versus adjacent normal esophageal tissues revealed significant differential expression of 756 genes. Among the most strongly up-regulated genes were keratin 17, immunoglobulin genes and ornithine decarboxylase 1. Genes that showed a great loss of expression included cysteine-rich secretory protein 3 and sciellin. We performed pathway and network analysis on significantly dysregulated genes using IPA to determine the overall biological impact of the widespread transcriptional aberration in African-American ESCC. We found significant dysregulation of genes encoding drug-metabolizing enzymes and stress response components of the NRF2- mediated oxidative damage pathway, potentially representing key genes in carcinogenesis in ESCC in African Americans. Loss of activity of drug metabolizing enzymes would confer increased sensitivity of esophageal cells to xenobiotics, such as alcohol and tobacco smoke, and may account for the high incidence and aggressiveness of ESCC in this ethnic group. A meta-analysis of ESCC expression profiles in our African American sample and those of several Asian samples determined genes in the NRF2 pathway are uniquely and significantly altered in African-American ESCC. Down-regulation of TP53 pathway components represented the most common feature in ESCC of all ethnic groups. Importantly, this analysis revealed a potential distinctive molecular property of African-American ESCC a widespread and prominent involvement of NRF2 pathway. Conclusion: Taken together, these findings highlight the remarkable interplay of genetic and environmental factors in the pathogenesis of African-American ESCC. Citation Format: Hayriye Verda Erkizan, Jack Lichy, Vincente Notario, Robert Wadleigh. African American esophageal squamous cell carcinoma expression profile reveals dysregulation of stress response and detox networks [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5566. doi:10.1158/1538-7445.AM2017-5566

Antagonizing miR-455-3p inhibits chemoresistance and aggressiveness in esophageal squamous cell carcinoma

BackgroundThe plasticity of cancer stem cells (CSCs)/tumor-initiating cells (T-ICs) suggests that multiple CSC/T-IC subpopulations exist within a tumor and that multiple oncogenic pathways collaborate to maintain the CSC/T-IC state. Here, we aimed to identify potential therapeutic targets that concomitantly regulate multiple T-IC subpopulations and CSC/T-IC-associated pathways.MethodsA chemoresistant patient-derived xenograft (PDX) model of human esophageal squamous cell carcinoma (ESCC) was employed to identify microRNAs that contribute to ESCC aggressiveness. The oncogenic effects of microRNA-455-3p (miR-455-3p) on ESCC chemoresistance and tumorigenesis were examined by in vivo and in vitro chemoresistance, tumorsphere formation, side-population, and in vivo limiting dilution assays. The roles of miR-455-3p in activation of the Wnt/β-catenin and transforming growth factor-β (TGF-β)/Smad pathways were determined by luciferase and RNA immunoprecipitation assays.ResultsWe found that miR-455-3p played essential roles in ESCC chemoresistance and tumorigenesis. Treatment with a miR-455-3p antagomir dramatically chemosensitized ESCC cells and reduced the subpopulations of CD90+ and CD271+ T-ICs via deactivation of multiple stemness-associated pathways, including Wnt/β-catenin and TGF-β signaling. Importantly, miR-455-3p exhibited aberrant upregulation in various human cancer types, and was significantly associated with decreased overall survival of cancer patients.ConclusionsOur results demonstrate that miR-455-3p functions as an oncomiR in ESCC progression and may provide a potential therapeutic target to achieve better clinical outcomes in cancer patients.

African-American esophageal squamous cell carcinoma expression profile reveals dysregulation of stress response and detox networks

BackgroundEsophageal carcinoma is the third most common gastrointestinal malignancy worldwide and is largely unresponsive to therapy. African-Americans have an increased risk for esophageal squamous cell carcinoma (ESCC), the subtype that shows marked variation in geographic frequency. The molecular architecture of African-American ESCC is still poorly understood. It is unclear why African-American ESCC is more aggressive and the survival rate in these patients is worse than those of other ethnic groups.MethodsTo begin to define genetic alterations that occur in African-American ESCC we conducted microarray expression profiling in pairs of esophageal squamous cell tumors and matched control tissues.ResultsWe found significant dysregulation of genes encoding drug-metabolizing enzymes and stress response components of the NRF2- mediated oxidative damage pathway, potentially representing key genes in African-American esophageal squamous carcinogenesis. Loss of activity of drug metabolizing enzymes would confer increased sensitivity of esophageal cells to xenobiotics, such as alcohol and tobacco smoke, and may account for the high incidence and aggressiveness of ESCC in this ethnic group. To determine whether certain genes are uniquely altered in African-American ESCC we performed a meta-analysis of ESCC expression profiles in our African-American samples and those of several Asian samples. Down-regulation of TP53 pathway components represented the most common feature in ESCC of all ethnic groups. Importantly, this analysis revealed a potential distinctive molecular underpinning of African-American ESCC, that is, a widespread and prominent involvement of the NRF2 pathway.ConclusionTaken together, these findings highlight the remarkable interplay of genetic and environmental factors in the pathogenesis of African-American ESCC.

Downregulation of MicroRNA-644a Promotes Esophageal Squamous Cell Carcinoma Aggressiveness and Stem Cell-like Phenotype via Dysregulation of PITX2.

We previously reported the oncogenic role of paired-like homeodomain 2 (PITX2) in esophageal squamous cell carcinoma (ESCC). In this study, we aimed to identify the miRNA regulators of PITX2 and the mechanism underlying the pathogenesis of ESCC. Using miRNA profiling and bioinformatics analyses, we identified miR-644a as a negative mediator of PITX2 in ESCC. A series of in vivo and in vitro assays were performed to confirm the effect of miR-644a on PITX2-mediated ESCC malignancy. ESCC cells and tissues expressed less miR-644a than normal epithelial controls. In patient samples, lower expression of miR-644a in ESCC tissues was significantly correlated with tumor recurrence and/or metastasis, such that miR-644a, PITX2, and the combination of the two were independent prognostic indicators for ESCC patient's survival (P < 0.05). Gain- and loss-of-function studies demonstrated that miR-644a inhibited ESCC cell growth, migration, and invasion in vitro and suppressed tumor growth and metastasis in vivo In addition, miR-644a dramatically suppressed self-renewal and stem cell-like traits in ESCC cells. Furthermore, the effect of upregulation of miR-644a was similar to that of PITX2 knockdown in ESCC cells. Mechanistic studies revealed that miR-644a attenuates ESCC cells' malignancy and stem cell-associated phenotype, at least partially, by inactivation of the Akt/GSK-3β/β-catenin signaling pathway through PITX2. Furthermore, promoter hypermethylation caused downregulation of miR-644a in ESCC. Downregulation of miR-644a plays an important role in promoting both aggressiveness and stem-like traits of ESCC cells, suggesting that miR-644a may be useful as a novel prognostic biomarker or therapeutic target for the disease. Clin Cancer Res; 23(1); 298-310. ©2016 AACR.

Computational analysis reveals microRNA-mRNA regulatory network in esophageal squamous cell carcinoma.

MicroRNAs (miRNAs) are known to regulate post-transcriptional gene expression. They are involved in carcinogenesis and tumor progression. The aim of this study was to explore the microRNA-mRNA regulatory network in esophageal squamous cell carcinoma (ESCC) using comprehensive computational approaches. In this study we have selected a total of 11 miRNAs from one previously reported study in ESCC. The mRNA targets of these miRNAs were predicted using various algorithms. The expression profiles of these mRNA targets were identified on DNA microarray experiment dataset across ESCC tissue samples. Based on the miRNA-mRNA regulatory relationships, the network was inferred. A total of 23 miRNA-mRNA regulatory interactions, with 11 miRNAs and 13 mRNA targets, were inferred in ESCC. The miRNA-mRNA regulatory network with increased confidence provides insights into the progression of ESCC and may serve as a biomarker for prognosis or the aggressiveness of ESCC. However, the results should be examined with further experimental validation.

Overexpression of SRC‐3 promotes esophageal squamous cell carcinoma aggressiveness by enhancing cell growth and invasiveness

Steroid receptor coactivator‐3 (SRC‐3), a transcriptional coactivator for nuclear receptors and other transcription factors, plays an important role in the genesis and progression of several cancers. However, studies investigated the role of SRC‐3 in esophageal squamous cell carcinomas (ESCCs) are limited, and the role of SRC‐3 in tumor progression remains unclear. We examined the expression of SRC‐3 in 8 ESCC cell lines and 302 human ESCC tissues by qPCR, Western blot, and immunohistochemistry. In addition, ESCC cell lines were subjected to proliferation and invasion assays, tumorigenicity assay, flow cytometry assay, qPCR, Western blot, and Chromatin Immunoprecipitation assay to investigate the role of SRC‐3 in cancer progression. SRC‐3 was overexpressed in 48% of cases and correlated with poor overall (P = 0.0076) and progression‐free (P = 0.0069) survival of surgically resected ESCC patient. Cox regression analysis revealed that SRC‐3 is an independent prognostic marker. Furthermore, we found that activation of insulin‐like growth factor (IGF)/AKT) was involved in the SRC‐3 on the cell growth and invasiveness in two ESCC cell lines, Eca109 and EC18 cells. SRC‐3 overexpression is clinically and functionally relevant to the progression of human ESCC, and might be a useful molecular target for ESCC prognosis and treatment.

Regulation of MMP13 by antitumor microRNA-375 markedly inhibits cancer cell migration and invasion in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive malignancies. Recently developed molecular targeted therapies are not available for patients with ESCC. After curative surgical resection, patients frequently suffer distant metastasis and recurrence. Exploration of novel ESCC metastatic pathways may lead to the development of new treatment protocols for this disease. Accordingly, we have sequentially identified microRNA (miRNA)-mediated metastatic pathways in several cancers. Our past studies of miRNA expression signatures have shown that microRNA-375 (miR-375) is frequently reduced in several types of cancers, including ESCC. In the present study, we aimed to investigate novel miR-375-mediated metastatic pathways in ESCC cells. The expression of miR-375 was downregulated in ESCC tissues, and ectopic expression of this miRNA markedly inhibited cancer cell migration and invasion, suggesting that miR-375 acted as an antimetastatic miRNA in ESCC cells. Our strategies for miRNA target searching demonstrated that matrix metalloproteinase 13 (MMP13) was directly regulated by miR-375 in ESCC cells. Overexpression of MMP13 was observed in ESCC clinical tissues, and the expression of MMP13 promoted cancer cell aggressiveness. Moreover, oncogenic genes, including CENPF, KIF14 and TOP2A, were shown to be regulated downstream of MMP13. Taken together, these findings demonstrated that the antitumor miR-375/oncogenic MMP13 axis had a pivotal role in ESCC aggressiveness. These results provide novel insights into the potential mechanisms of ESCC pathogenesis.

Integrin expression in esophageal squamous cell carcinoma: loss of the physiological integrin expression pattern correlates with disease progression.

The integrins are a family of heterodimeric transmembrane signaling receptors that mediate the adhesive properties of epithelial cells affecting cell growth and differentiation. In many epithelial malignancies, altered integrin expression is associated with tumor progression and often correlates with unfavorable prognosis. However, only few studies have investigated the role of integrin expression in esophageal squamous cell carcinoma (ESCC). Using a novel quantifying immunofluorescence-staining assay, we investigated the expression of the integrins α2β1, α3β1, α6β1, and α6β4 in primary ESCC of 36 patients who underwent surgical resection. Magnitude and distribution of expression were analyzed in primary tumor samples and autologous esophageal squamous epithelium. The persistence of the physiologically polarized expression of the subunits α6, β1, and β4 in the tumor tissue was significantly associated with prolonged relapse-free survival (p = 0.028, p = 0.034, p = 0.006). In contrast, patients with reduced focal α6 expression at the tumor invasion front shared a significantly shortened relapse-free survival compared to patients with strong α6 expression at their stromal surfaces, as it was regularly observed in normal esophageal epithelium (p = 0.001). Multivariate regression analysis identified the maintenance of strong α6 immunoreactivity at the invasion front as an independent prognostic factor for increased relapse-free and disease-specific survival (p = 0.003; p = 0.003). Our findings suggest that alterations in both pattern and magnitude of integrin expression may play a major role in the disease progression of ESCC patients. Particularly, the distinct expression of the integrins α6β4 and α6β1 at the invasion front as well as the maintenance of a polarized integrin expression pattern in the tumor tissue may serve as valuable new markers to assess the aggressiveness of ESCC.