Proliferation Of ESCC Research Articles

Fbxw8 protein promotes the proliferation and metastasis of ESCC

Objective: This article plans to investigate Fbxw8 protein expression in esophageal squamous cell carcinoma tissues. And its effect on the proliferation ability of esophageal squamous cancer KYSE70 cells is analyzed. Methods: We can detect Fbxw8 protein expression in esophageal squamous cell carcinoma tissues with IHC and analysis the expression with the sex, age, lymph node metastasis and tumor stage. We alsodetect the proliferation of KYSE70 cells with CCK 8. Result: Most esophageal squamous carcinoma tissues highly expressed Fbxw8 protein, and the expression was positively correlated with lymph node metastasis and tumor staging (P < 0.05). Fbxw8 siRNAs can significantly cut FBXW8 protein expression, while lowering the cell cycle related protein expression of Cyclin A, Cyclin B1, and inhibiting KYSE70 cell proliferation. Conclusion: Fbxw8 protein highly expressed in esophageal squamous carcinoma tissues, and closely related to tumor staging. Targeted inhibition Fbxw8 protein expression could significantly inhibit the proliferation of esophageal squamous carcinoma cells.

P11 KRT17 PROMOTES PROLIFERATION AND METASTASIS OF ESOPHAGEAL SQUAMOUS CELL CARCINOMA LEADING TO POOR PROGNOSIS

Abstract Background Keratin17(KRT17), as a multifunction cytoskeletal protein, associated with a multitudinous of biological processes, including cell proliferation, migration, and invasion. Previously, we have found overexpression of KRT17 genes in ESCC tissues. Currently, published kinds of researches claimed KRT17 is engaged in the tumorigenesis and progression of multiple cancers. However, the prognostic significance of KRT17 in ESCC patients and its effects in ESCC progression remain indistinct. Methods We verified the expression of KRT17 in ESCC tissues by Western blotting and q-PCR. And then the immunochemistry was performed to investigate the KRT17 expression in 64 ESCC tissue specimens. KRT17 upregulated, and knockdown EC9706 and Eca109 cells were constructed by Lentivirus overexpression and CRISPR-Cas9. The function of KRT17 in ESCC proliferation and metastasis were studied by CCK-8 assays, plate colony formation assays, trans-well and wound healing in vitro. Results overexpression of KRT17 was verified in 12 pairs of fresh ESCC tissues. We found that the expression of KRT17 was remarkably positively correlated with the depth of invasion (T) (P<0.05), lymph node metastasis (N) (P<0.05), tumor node metastasis (TNM) stage (P<0.0001). High expression of KRT17 predicted a lower 5-year survival rate(P<0.001) of ESCC patients. We verified the KRT17 malignant behavior of ESCC in vitro. We conformed proliferation and migration capacity of ESCC were significantly enhanced by overexpression of KRT17 but compromised when KRT17 was knocked down. Collectively, it supported that the increased expression of KRT17 promoted proliferation and migratory phenotype of ESCC. Conclusion The breakthrough indicated that the upregulation of KRT17 in ESCC is closely related to malignant progression, our findings revealed that KRT17 plays a vital role in facilitating proliferation and metastasis, and could be used as a novel predictive biomarker, thus providing a novel diagnosis and therapeutic target for ESCC patients. Keywords: Molecular target ESCC Keratin17 Therapeutic target

P8 KERATIN 17 PROMOTES CELLS PROLIFERATION AND METASTASIS IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA

Abstract Background Keratin17(KRT17), as a multifunction cytoskeletal protein, associated with a multitudinous of biological processes, including cell proliferation, migration, and invasion. Previously, we have found up-expression of KRT17 genes in ESCC tissues. Currently, published kinds of researches claimed KRT17 is engaged in the tumorigenesis and progression of multiple cancers. However, the prognostic significance of KRT17 in ESCC patients and its effects in ESCC progression remains indistinct. Methods We verified the expression level of KRT17 in ESCC tissues by Western blotting and q-PCR and constructed KRT17 upregulated and knockdown EC9706 and Eca109 cells by Lentivirus overexpression and CAS9. The function of KRT17 in ESCC proliferation and metastasis were studied in vitro. We performed CCK-8 assays and plate colony formation assays and the plate colony formation assays to explore the effect of KRT17 on the proliferation, Moreover, the trans-well migration chamber experiment and wound healing test was taken to reveal the impact of KRT17 on migration capabilities. Results We verified that KRT17 is overexpressed in ESCC tissues and the role of KRT17 in the malignant behavior of ESCC in vitro and vivo . In CCK-8 assays and plate colony formation assays. We conformed proliferation capacity was notably enhanced by overexpression of KRT17 but compromised when KRT17 was knocked out. On the other hand, we used the plate colony formation assays to explore the effect of KRT17 on the proliferation of ESCC cells. KRT17 up-regulated significantly strengthened the proliferation while the KRT17 knockdown was weakened. To reveal the impact of KRT17 on migration, the trans-well migration chamber experiment was used to compare the role of down-regulation and up-regulation of KRT17 in migration; the results demonstrated that reducing expression of KRT17 significantly blocked the movement of ESCC cells as compared with the empty vector control. Otherwise, it has been confirmed that the cell migration ability employs a wound healing/scratch test because wound closure is generally a measure of cell motility. The results showed KRT17 overexpression notably reinforced the mobility of ESCC cells compared with the empty groups, whereas KRT17 knockdown cells prolonged the time to close the injury compared to blank groups. Collectively, these discoveries support that the increased expression of KRT17 in ESCC cells promoted a more proliferation and migratory phenotype in vitro. Conclusion The breakthrough indicated that the up-regulation of KRT17 in ESCC is closely related to malignant progression. Our data confirmed that KRT17 plays an essential role in reinforcing proliferation and metastasis in ESCC. Thence, KRT17 may serve as a significant molecular target for the diagnosis and therapy of ESCC.

LncRNA ZEB1-AS1 down-regulation suppresses the proliferation and invasion by inhibiting ZEB1 expression in oesophageal squamous cell carcinoma.

Multiple studies have unveiled that long non‐coding RNAs (lncRNAs) play a pivotal role in tumour progression and metastasis. However, the biological role of lncRNA ZEB1‐AS1 in oesophageal squamous cell carcinoma (ESCC) remains under investigation, and thus, the current study was to investigate the functions of ZEB1‐AS1 in proliferation and invasion of ESCC. Here, we discovered that ZEB1‐AS1 and ZEB1 were markedly up‐regulated in ESCC tissues and cells relative to their corresponding normal control. ZEB1‐AS1 and ZEB1 overexpressions were both related to TNM staging and lymph node metastasis as well as poor prognosis in ESCC. The hypomethylation of ZEB1‐AS1 promoter triggered ZEB1‐AS1 overexpression in ESCC tissues and cells. In addition, ZEB1‐AS1 knockdown mediated by siRNA markedly suppressed the proliferation and invasion in vitro in EC9706 and TE1 cells, which was similar with ZEB1 siRNA treatment, coupled with EMT alterations including the up‐regulation of E‐cadherin level as well as the down‐regulation of N‐cadherin and vimentin levels. Notably, ZEB1‐AS1 depletion dramatically down‐regulated ZEB1 expression in EC9706 and TE1 cells, and ZEB1 overexpression obviously reversed the inhibitory effects of proliferation and invasion triggered by ZEB1‐AS1 siRNA. ZEB1‐AS1 shRNA evidently inhibited tumour growth and weight, whereas ZEB1 elevation partly recovered the tumour growth in ESCC EC9706 and TE1 xenografted nude mice. In conclusion, ZEB1‐AS1 overexpression is tightly involved in the development and progression of ESCC, and it exerts the antitumour efficacy by regulating ZEB1 level in ESCC.

Abstract 4299: Targeting AKT with oridonin inhibits growth of esophageal squamous cell carcinoma in vitro and patient-derived xenografts in vivo

Abstract Esophageal cancer is the sixth leading cause of cancer death worldwide and has low survival rates with poor prognosis. Hyperactivation of AKT has been reported to modulate cell growth, survival, and gene expression in various solid tumors. Oridonin, an inflammatory medical and diterpenoid compound isolated from Rabdosia rubescens, has exhibited various pharmacologic and physiologic properties, including antitumor, antibacterial, and anti-inflammatory effects. In this study, we aimed to investigate the effects of oridonin on the proliferation and growth of ESCC and to elucidate its underlying mechanisms of action. We found that oridonin is an inhibitor of AKT and induces cell cycle arrest and apoptosis in ESCC cells and attenuates growth of patient derived xenograft (PDX) tumors in vivo by interfering with AKT signaling pathways through MTT assay, anchorage-independent cell growth assay and Cell cycle and apoptosis analyses. AKT was developed as a dierctly target of oridonin by in vitro kinase assay, ex vivo and in vitro pull-down assay and computational docking model. Mechanistically, oridonin diminished the phosphorylation and activation of AKT and supressed the downstream of p-GSK-3β, p-mTOR and NF-kB activity. Moreover, a combination of oridonin and 5-fluorouracil or cisplatin (clinical chemotherapeutic agents) enhanced the inhibition of ESCC cell growth. Overall, our study suggests that oridonin can inhibit progression of ESCC tumors in vitro and in vivo by suppressing AKT signaling through its direct targeting of AKT. Thus, this study might provide useful information in the clinical application of oridonin for ESCC chemotherapy. Citation Format: Mengqiu Song, Ran Zhao, Hua Xie, Hanyong Chen, Kangdong Liu, Ann M. Bode, Mee-Hyun Lee, Zigang Dong. Targeting AKT with oridonin inhibits growth of esophageal squamous cell carcinoma in vitro and patient-derived xenografts in vivo [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 4299.

Abstract 509: Upregulation of miR-424 promotes cell cycle progression and cell proliferation in esophageal squamous cell carcinoma

Abstract Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive malignancies of the gastrointestinal tract. miR-424 was one of the most significantly upregulated miRNAs in the ESCCs compared with normal esophageal tissues (NEs) identified by our previous miRNA arrays. The effects of miR-424 in tumor development and progression are conflicting according to previous reports till now. Here, we characterized the role of miR-424 in ESCC cell cycle progression and proliferation regulation. Expression of miR-424 was determined using qRT-PCR analysis. Cell proliferation assay by CCK-8 and the xenograft tumor model were used to determine ESCC proliferation in vitro and in vivo. Cell cycle distribution was analyzed by flow cytometry after synchronizing and releasing cells from G0/G1 phase, the onset of S-phase, or mitosis. Targets of miR-424 were validated using luciferase assays. The presence of E2F1-binding sites in the putative promoter of miR-424 gene was investigated using Chromatin immunoprecipitation (ChIP). As a result, miR-424 expression was elevated in ESCC specimens than in their normal counterparts. High miR-424 expression was a predictor of poor survival for Stage II-III ESCC patients undergoing surgery. Consistent with clinical data, knockdown miR-424 function in ESCC cells remarkably suppressed ESCC proliferation in vitro and tumor formation in mice model, while opposite results were observed after overexpressing miR-424. Cell cycle analyses revealed that miR-424 knockdown negatively regulates both G1/S and G2/M transition in ESCC cells, which were mediated by two newly identified miR-424 targets PRKCD and WEE1, respectively. During G1/S transition, increased level of PRKCD protein in KYSE410-miRZip-424 cells resulted in increased activation of PRKCD and downstream p38MAPK and JNK, which enhanced the stability of p21Cip1 protein. p21Cip1 proceeded to block CDK2 activation during G1/S transition. On the other hand, level of CDC2 phosphorylation at Tyr15, the inactive state of CDC2, was increased as a result of increased WEE1 expression by miR-424 knockdown when cells were at the G2/M border. Further evaluation of miR-424 expression in various cell cycle phases showed that miR-424 expression increased along with pri-miR-424 and pre-miR-424 when cell cycle progressed from G0/G1- to S-phase in ESCC cells. ChIP results demonstrated that G1/S transcriptional activating regulator E2F1 was enriched in regions of the putative miR-424 promoter containing the predicted E2F1-binding sites. Here, our results proved that miR-424 functions as an onco-miRNA in ESCCs. Activation of E2F1 when ESCC cells at the G1/S border induced the transcription of miR-424, which in turn targeted the expression of key genes to promote cell cycle progression and cell proliferation of ESCCs. Citation Format: Jing Wen, Xiuying Xie, Jianhua Fu. Upregulation of miR-424 promotes cell cycle progression and cell proliferation in 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 509.

Abstract 3328: Association between LOX expression, LINE-1 DNA methylation and prognosis in esophageal cancer

Abstract Background: Abnormal function of human body enzymes and epigenetic alterations such as DNA methylation lead to human carcinogenesis. Lysyl oxidase (LOX) is an extracellular matrix remodeling enzyme and its upregulation has been associated with poor prognosis in various cancers. LINE-1 methylation is considered as a surrogate marker of global DNA methylation and is attracting interest as a good predictor of cancer prognosis. Despite LOX involvement in tumor proliferation, migration and invasion in other types of tumors, the importance of LOX enzyme and its epigenetic regulation in pathogenesis of esophageal cancer has yet to be fully elucidated. Therefore, we constructed this study to examine the correlations between LOX expression, LINE-1 DNA methylation and the clinical pathological profile in patients with esophageal cancer. Methods: mRNA expression array analysis was done using tissue biopsy and public array data. We then examined LOX expression in esophageal cancer using tissue biopsy by Real time PCR.LOX expression was evaluated in 284 surgically resected ESCC by immunohistochemistry, and the correlation was analyzed with LINE-1 methylation, obtained previously with pyrosequencing. In vitro we looked on LOXs role in tumor progression by growth and invasion assay using ESCC cell lines. Results: Our microarray data reveled that LOX was the highly upregulated gene in hypo methylated tumors (fold change>2X). In tissue biopsies LOX was significantly higher in tumor part compared to normal epithelium. In immunohistological study, LOX was identified in tumor cytoplasm. High LOX expression correlated with decrease in Overall survival, cancer-specific survival and disease-free survival. (P <0.001**). Moreover, significant association with tumor size, invasion depth, tumor stage and lymph node metastases was also observed. In multivariate analysis LOX expression. and tumor depth were an independent prognostic factor. Nevertheless, high LOX expression correlated to lower methylation levels (P=0.01). Invitro, we observed upstream of LOX expression by 5-AZA treatment but LOX knockdown had no influence in methylation levels. LOX knockdown suppressed growth in both TE6 and TE11 and reduced invasion and migration ability in TE 11 cell line. Conclusions: Our findings show LOX is regulated epigenetically by genome wide hypomethylation and promote invasion and proliferation of ESCC. Hence, LOX can serve as a prognostic biomarker in ESCC patients and therapeutically targeting LOX could reverse progression of esophageal cancer. 2269/2600 characters Citation Format: Rebecca Tauzen Kalikawe, Yoshifumi Baba, Miyake Keisuke, Yagi Taisuke, Yuki Kitano, Sawayama Hiroshi, Hoyoshi Yukiharu, Iwatsuki Maasaki, Miyamoto Yuji, Yoshida Naoya, Ishimoto Takatsugu, Hideo Baba. Association between LOX expression, LINE-1 DNA methylation and prognosis in esophageal cancer [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 3328.

MicroRNA and target mRNA selection through invasion and cytotoxicity cell modeling and bioinformatics approaches in esophageal squamous cell carcinoma

This study analyzed microRNA(miRNA) and mRNA expression profiles and investigated the biological characteristics of ESCC by using invasion and cytotoxicity cell models. miRNA profiles were evaluated through miRNA microarray. Transwell chamber and nedaplatin(NDP) were used to construct invasion and cytotoxicity cell models. Invasion Transwell and cytotoxicity assays were performed to examine the invasiveness and proliferation in the cell models. Functional miRNAs were selected from dysregulated miRNAs through qRT-PCR. Biometric Research Program(BRB)-array tools, Cytoscape plugins, and DAVID were utilized to find potential mRNAs targeted by these two miRNAs between ESCC and paired normal adjacent tissues. Our microarray obtained 11 dysregulated miRNAs expressed in three paired ESCC samples from Kazakhs (ethnicity in Northwestern China). qRT-PCR demonstrated the miRNA expression in the invasion and cytotoxicity cell models. miR‑652-5p and miR‑21‑5p exhibited a consistent expression level in the microarray and cell models. Bioinformatics revealed that the potential targets of PLD1, MSH2, STC1, and DSG1 might be involved in ESCC invasion and proliferation. Cell models with bioinformatics approaches may help distinguish functional genes. miR‑652-5p, miR‑21‑5p, and their potential target genes may participate in ESCC development and metastasis.

Antitumor effect of metformin in esophageal cancer: In vitro study

Recent studies suggest that metformin, which is a member of the biguanide family and commonly used as an oral anti-hyperglycemic agent, may reduce cancer risk and improve prognosis of numerous types of cancer. However, the mechanisms underlying the antitumor effect of metformin on esophageal cancer remain unknown. The goal of the present study was to evaluate the effects of metformin on the proliferation of human ESCC in vitro, and to study changes in the expression profile of microRNAs (miRNAs), since miRNAs have previously been associated with the antitumor effects of metformin in other human cancers. The human ESCC cell lines T.T, KYSE30 and KYSE70 were used to study the effects of metformin on human ESCC in vitro. In addition, we used miRNA array tips to explore the differences between miRNAs in KYSE30 cells with and without metformin treatment. Metformin inhibited the proliferation of T.T, KYSE30 and KYSE70 cells in vitro. Metformin blocked the cell cycle in G0/G1 in vitro. This blockade was accompanied by a strong decrease of G1 cyclins, especially cyclin D1, as well as decreases in cyclin-dependent kinase (Cdk)4, Cdk6 and phosphorylated retinoblastoma protein (Rb). In addition, the expression of miRNAs was markedly altered with the treatment of metformin in vitro. Metformin inhibited the growth of three ESCC cell lines, and this inhibition may have involved reductions in cyclin D1, Cdk4 and Cdk6.

* LNA-Modified oligonucleotides targeting mRNA of dicer in pegylated colloidal nanoparticles with linked ABS against CD44 eradicate esophageal cancer stem cells (ECSC) by induction of PCD after inhibition of oncomir machinery

IntroductionMetastatic oesophageal squamous cell Ca (ESCC) is incurable due to chemoresistance caused by cancer stem cells due to overexpression of oncomirs which upregulate oncogenes and hypermethylation in CpG islands which...

Bile Acid Promotes the Proliferation of Squamous Cell Carcinoma of the Esophagus, Independent of its Inducing COX-2 Expression

We have reported G1 progression and cyclooxygenase-2 (COX-2) induction during carcinogenesis of the squamous epithelium of the esophagus. As bile acid induces COX-2 expression and promotes carcinogenesis in the digestive tract, we investigated the effect of bile acid on the proliferation of squamous cell carcinoma of the esophagus (ESCC). MTT assay, Western blot analysis of COX-2 and cell cycle-related molecules in the G1 phase (Rb, CDC25A, cyclin D1), and CDK2 kinase assay were performed on chenodeoxycholic acid (CDCA) exposure to ESCC cell lines (TE2R, TE3, TE13, TE15). In the presence of gradient bile acid concentration (up to 100 microm), growth of ESCC cell lines was stimulated at a low concentration (maximally at 20-30 microm), but suppressed at a higher concentration. Only a low dose of bile acid induced the expression of cyclin D1 and CDC25A and showed high Rb phosphorylation and high CDK2 kinase activity. In contrast, bile acid progressively induced COX-2 expression in a dose-dependent manner, regardless of its biphasic effects on cell proliferation, and a COX-2 specific inhibitor (JTE-522) did not suppress growth stimulation by a low dose of bile acid. Bile acid at a low dose stimulates the proliferation of ESCC by inducing G1-regulating molecules. However, COX-2 expression, which is also induced by bile acid, does not affect cell proliferation. Further work is needed to elucidate its role in carcinogenesis.