Esophageal Squamous Cell Carcinoma Cell Proliferation Research Articles

Exosomal and intracellular miR-320b promotes lymphatic metastasis in esophageal squamous cell carcinoma

Cancer-cell-released exosomal microRNAs (miRNAs) are important mediators of cell-cell communication in the tumor microenvironment. In this study, we sequenced serum exosome miRNAs from esophageal squamous cell carcinoma (ESCC) patients and identified high expression of miR-320b to be closely associated with peritumoral lymphangiogenesis and lymph node (LN) metastasis. Functionally, miR-320b could be enriched and transferred by ESCC-released exosomes directly to human lymphatic endothelial cells (HLECs), promoting tube formation and migration in vitro and facilitating lymphangiogenesis and LN metastasis in vivo as assessed by gain- and loss-of-function experiments. Furthermore, we found programmed cell death 4 (PDCD4) as a direct target of miR-320b through bioinformatic prediction and luciferase reporter assay. Re-expression of PDCD4 could rescue the effects induced by exosomal miR-320b. Notably, the miR-320b-PDCD4 axis activates the AKT pathway in HLECs independent of vascular endothelial growth factor-C (VEGF-C). Moreover, overexpression of miR-320b promotes the proliferation, migration, invasion, and epithelial-mesenchymal transition progression of ESCC cells. Finally, we demonstrate that METTL3 could interact with DGCR8 protein and positively modulate pri-miR-320b maturation process in an N6-methyladenosine (m6A)-dependent manner. Therefore, our findings uncover a VEGF-C-independent mechanism of exosomal and intracellular miR-320b-mediated LN metastasis and identify miR-320b as a novel predictive marker and therapeutic target for LN metastasis in ESCC.

FoXA2 promotes esophageal squamous cell carcinoma progression by ZEB2 activation

BackgroundIt has been reported that Forkhead transcription family member (FOXA2) regulates esophageal squamous cell carcinoma (ESCC) progression. However, the specific mechanism, by which FOXA2 promotes ESCC malignant progression, remains unclear.Materials and methodsQRT-PCR and western blotting were applied to measure FOXA2 expression in ESCC tissues, while CCK-8 assay and Transwell assays were used to investigate the effect of FOXA2 on ESCC. Luciferase reporter assay, followed by fast chromatin immunoprecipitation (ChIP) assay, was used to study the relationship between FOXA2 and ZEB2.ResultsFOXA2 was significantly increased in ESCC tissues, when compared to normal tissues. Moreover, high expression of FOXA2 was also found in ESCC cells. Knockdown of FOXA2 inhibited ESCC cell proliferation, invasion, and migration. Mechanically, FOXA2 was verified to regulate ZEB2 expression at transcription level. Moreover, ZEB2 reversed the inhibitory effect of FOXA2 on ESCC proliferation, invasion, and migration. The relationship between ZEB2 and FOXA2 in ESCC tissues was negative.ConclusionsThese results indicate that FOXA2 plays a critical role in ESCC progression and may become a potential candidate target for ESCC treatment.

LINC00514 promotes lipogenesis and tumor progression in esophageal squamous cell carcinoma by sponging miR‑378a‑5p to enhance SPHK1 expression.

Increasing evidence has demonstrated that long non-coding RNAs serve pivotal roles in tumor development, progression, metastasis and metabolism. However, to the best of our knowledge, the roles and molecular mechanisms of long intergenic nonprotein-coding RNA 00514 (LINC00514) in esophageal squamous cell carcinoma (ESCC) remain unknown. The present study found that LINC00514 and sphingosine kinase 1 (SPHK1) were both upregulated in ESCC tissues and cells, and their high expression levels were closely associated with Tumor-Node-Metastasis stage, lymph node metastasis and poor prognosis of patients with ESCC. Functionally, knockdown of LINC00514 inhibited cell proliferation and invasion, and led to the downregulation of lipogenesis-related proteins, including SPHK1, fatty acid synthase, acetyl-coenzyme (Co)A carboxylase α and stearoyl-CoA desaturase 1, whereas LINC00514 overexpression promoted cell proliferation and invasion in ESCC KYSE150 and KYSE30 cells, and upregulated expression of lipogenesis-related proteins. Mechanistically, LINC00514 functioned as a competing endogenous RNA by sponging microRNA (miR)-378a-5p, resulting in the upregulation of SPHK1, which was accompanied by the activation of lipogenesis-related pathways, to promote ESCC cell proliferation and invasion. Taken together, these findings suggest that LINC00514 may participate in ESCC lipogenesis, and targeting the LINC00514/miR-378a-5p/SPHK1 signaling axis may be a novel and promising therapeutic strategy for management of patients with ESCC.

Reduced tissue and serum resistin expression as a clinical marker for esophageal squamous cell carcinoma.

Esophageal cancer is one of the most common malignancies and leading cause of cancer-associated mortality worldwide. However, the molecular mechanisms underlying esophageal cancer progression and the development of clinical tools for effective diagnosis remain unclear. Resistin, which was originally identified as an adipose tissue-secretory factor, has been associated with obesity-related diseases, including certain types of cancer. Thus, the present study aimed to investigate the expression levels of resistin in tissue and serum specimens from patients with esophageal squamous cell carcinoma (ESCC) to determine the potential biological effects of resistin on ESCC cells. The results demonstrated that both tissue and serum resistin levels were significantly lower in patients with ESCC compared with healthy controls. In addition, resistin expression was positively associated with the body mass index of patients with ESCC. In vitro studies revealed that resistin inhibited the migratory ability of ESCC cells, while having no effect on ESCC cell proliferation. Taken together, these results suggest that resistin may have the potential to be developed into a clinical marker for ESCC. However, further studies are required to investigate resistin receptor expression and determine the potential involvement of resistin-associated biological pathways, which may provide insight for future development of targeted therapies for resistin-mediated ESCC.

Discovery, Optimization, and Structure-Activity Relationship Study of Novel and Potent RSK4 Inhibitors as Promising Agents for the Treatment of Esophageal Squamous Cell Carcinoma.

Ribosomal S6 protein kinase 4 (RSK4) was identified to be a promising target for the treatment of esophageal squamous cell carcinoma (ESCC) in our previous research, whose current treatments are primarily chemotherapy and radiotherapy due to the lack of targeted therapy. However, few potent and specific RSK4 inhibitors are reported. In this study, a series of 1,4-dihydro-2H-pyrimido[4,5-d][1,3]oxazin-2-ones derivatives were designed and synthesized as novel and potent RSK4 inhibitors. Compound 14f was identified with potent RSK4 inhibitory activity both in vitro and in vivo. 14f significantly inhibited the proliferation and invasion of ESCC cells in vitro with IC50 values of 0.57 and 0.98 μM, respectively. It dose dependently inhibited the phosphorylation of RSK4 downstream substrates while exerting little effect on the substrates of RSK1-3 in ESCC cells. The markedly suppressed tumor growth and no observed toxicity to main organs in the ESCC xenograft mouse model suggested 14f to be a promising RSK4-targeting agent for ESCC treatment.

M6A demethylase ALKBH5 suppression contributes to esophageal squamous cell carcinoma progression.

Esophageal squamous cell carcinoma (ESCC) is a highly malignant gastrointestinal cancer with a high recurrence rate and poor prognosis. Although N6-methyladenosine (m6A), the most abundant epitranscriptomic modification of mRNAs, has been implicated in several cancers, little is known about its participation in ESCC progression. We found reduced expression of ALKBH5, an m6A demethylase, in ESCC tissue specimens with a more pronounced effect in T3-T4, N1-N3, clinical stages III–IV, and histological grade III tumors, suggesting its involvement in advanced stages of ESCC. Exogenous expression of ALKBH5 inhibited the in vitro proliferation of ESCC cells, whereas depletion of endogenous ALKBH5 markedly enhanced ESCC cell proliferation in vitro. This suggests ALKBH5 exerts anti-proliferative effects on ESCC growth. Furthermore, ALKBH5 overexpression suppressed tumor growth of Eca-109 cells in nude mice; conversely, depletion of endogenous ALKBH5 accelerated tumor growth of TE-13 cells in vivo. The growth-inhibitory effects of ALKBH5 overexpression are partly attributed to a G1-phase arrest. In addition, ALKBH5 overexpression reduced the in vitro migration and invasion of ESCC cells. Altogether, our findings demonstrate that the loss of ALKBH5 expression contributes to ESCC malignancy.

LncRNA SNHG17 regulates cell proliferation and invasion by targeting miR-338-3p/SOX4 axis in esophageal squamous cell carcinoma

Small nucleolar RNA host gene 17 (SNHG17), a novel functional long noncoding RNA, has been demonstrated to play an essential role in the oncogenesis of several tumors. However, for esophageal squamous cell carcinoma (ESCC) the expression pattern and detailed function of SNHG17 are largely unknown. Hence, we conducted this study to explore potential roles and underlying oncogenic mechanisms for SNHG17 in ESCC progression. Results demonstrated SNHG17 to be markedly upregulated in ESCC. Knockdown of SNHG17 significantly suppressed ESCC cell proliferation, invasion, and epithelial–mesenchymal transition in vitro and tumor growth in vivo. Online database software analysis found miR-338-3p to interact with SNHG17 with the level of miR-338-3p negatively correlated with SNHG17 levels in ESCC samples. Further, miR-338-3p was found to directly target SRY-box transcription factor 4 (SOX4) in ESCC cells. Mechanistic analysis suggested that SNHG17 acts as an endogenous “sponge” competing with miR-338-3p to regulate SOX4, thereby promoting tumor progression. These results suggest that these molecular interactions may be potential therapeutic targets for ESCC.

Effect of the acid suppressor omeprazole on the proliferation, migration, invasion and cell cycle of esophageal squamous cell carcinoma cells via the aryl hydrocarbon receptor pathway.

Esophageal cancer is a malignant tumor type with one of the highest mortality rates worldwide. The aryl hydrocarbon receptor (AHR), which has been investigated in recent years, has been confirmed to be associated with the occurrence and development of esophageal cancer. AHR has a variety of different ligands, which regulate its activity following binding. The widely known acid inhibitor omeprazole (OME) also affects AHR and its downstream proteins (such as the cytochrome P450 family) by non-ligand binding; however, the mechanisms have remained to be fully elucidated. Therefore, the aim of the present study was to investigate the role of OME in esophageal squamous cell carcinoma (ESCC), whether the mechanism proceeds via the AHR pathway and how OME regulates AHR to affect the occurrence and development of esophageal carcinoma. The AHR-selective regulator OME was used to treat the ESCC cell lines TE1 and KYSE150. Western blot analysis was used to verify the effect of OME on AHR and proliferating cell nuclear antigen (PCNA) protein expression levels, while Cell Counting Kit (CCK)-8, wound-healing and Transwell assays were used to determine the proliferation, migration and invasion of the ESCCs, respectively, following treatment with OME. In addition, flow cytometry was used to investigate the cell cycle distribution of the ESCCs following incubation with OME. AHR was highly expressed in the ESCCs and following treatment with OME, the protein expression levels of AHR and PCNA were downregulated. The CCK-8 assay indicated that the proliferation of the ESCCs was also reduced following treatment with OME. Furthermore, flow cytometry revealed a notable block of the cells in G1/G0 phase, while the results of the wound-healing and Transwell assays respectively suggested that cell migration and invasion were reduced. In conclusion, OME inhibited the proliferation, migration and invasion of ESCC cells and blocked the cell cycle via the AHR pathway, which may provide a therapeutic effect on esophageal squamous cell cancer.

Hsa_circ_0014879 regulates the radiosensitivity of esophageal squamous cell carcinoma through miR-519-3p/CDC25A axis.

Circular RNAs (circRNAs) play critical roles in regulating the radiosensitivity of various cancers, including esophageal squamous cell carcinoma (ESCC). This research aimed to explore the role and potential mechanism of hsa_circ_0014879 in regulating ESCC radioresistance. The levels of hsa_circ_0014879, microRNA-519-3p (miR-519-3p) and cell division cycle 25A (CDC25A) were measured using quantitative real-time PCR or western blot. Cell proliferation was evaluated by colony formation assay. Cell migration and invasion were assessed by transwell and scratch assays. The levels of epithelial-mesenchymal transition (EMT)-related proteins were detected by western blot. Xenograft assay was used to analyze the effect of hsa_circ_0014879 on radiosensitivity in vivo. The binding relationship among hsa_circ_0014879, miR-519-3p and CDC25A was confirmed by dual-luciferase reporter assay. Hsa_circ_0014879 and CDC25A were upregulated, whereas miR-519-3p was downregulated in radio-resistant ESCC tissues and cells. Depletion of hsa_circ_0014879 suppressed the proliferation, migration and invasion of radio-resistant ESCC cells. Hsa_circ_0014879 knockdown elevated radiosensitivity of radio-resistant cells by modulating miR-519-3p. Moreover, miR-519-3p enhanced the radiosensitivity of radio-resistant cells by targeting CDC25A. Also, hsa_circ_0014879 upregulated CDC25A via sponging miR-519-3p. Hsa_circ_0014879 silencing enhanced the radiosensitivity of ESCC via regulating the miR-519-3p/CDC25A pathway.

GFI1 promotes the proliferation and migration of esophageal squamous cell carcinoma cells through the inhibition of SOCS1 expression.

Growth factor-independent 1 (GFI1) has been reported to serve a vital role in hematopoietic development. However, the function and molecular mechanism of GFI1 in esophageal squamous cell carcinoma (ESCC) remains unknown. In the present study, the biological functions and the molecular mechanism of the effects of GFI1 in ESCC were analyzed. The results demonstrated that GFI1 expression levels were significantly upregulated in ESCC compared with those in normal esophageal tissues. Knockdown of GFI1 using small interfering RNA suppressed ESCC cell proliferation and migration. Furthermore, GFI1 enhanced STAT3 and NF-κB signaling by inhibiting the expression of suppressor of cytokine signaling 1 (SOCS1) in ESCC cells. Taken together, the results of the present study demonstrated that GFI1 promoted the proliferation and migration of ESCC cells via inhibition of SOCS1 expression. These results suggested that GFI1 may be a valuable target for ESCC therapy.

Linc00941 regulates esophageal squamous cell carcinoma via functioning as a competing endogenous RNA for miR-877-3p to modulate PMEPA1 expression.

Esophageal squamous cell carcinoma (ESCC) represents one of the most common malignancies and is the fifth leading cause of cancer-related deaths. Long intergenic non-coding RNAs (lincRNAs) have been suggested to be dysregulated in various types of cancers, and a growing number of lincRNAs have been implicated to be functional in the ESCC progression. In this study, we examined the role of linc00941 in the ESCC progression and explored the underlying molecular mechanisms. The bioinformatics analysis identified the up-regulation of linc00941 in the ESCC tissues. Further in vitro studies showed that linc00941 was up-regulated in ESCC cell lines. The loss-of-function studies demonstrated that linc00941 knockdown suppressed ESCC cell proliferation, invasion and migration, and also suppressed the in vivo tumor growth. Furthermore, bioinformatics prediction along with luciferase reporter assay and RNA immunoprecipitation assay implied that linc00941 acted as a competing endogenous RNA for miR-877-3p, and linc00941 regulated ESCC cell progression via at least targeting miR-877-3p. Subsequently, miR-877-3p targeted prostate transmembrane protein, androgen induced 1 (PMEPA1) 3’ untranslated region and repressed PMEPA1 expression in ESCC cells; overexpression of PMEPA1 attenuated the inhibitory effects of linc00941 knockdown on the ESCC cell progression. Linc00941 knockdown suppressed epithelial-mesenchymal transition (EMT) via targeting miR-877-3p/PMEPA1 axis in ESCC cells. In conclusion, our results indicated the oncogenic role of linc00941 in ESCC, and knockdown of linc00941 suppressed ESCC cell proliferation, invasion, migration and EMT via interacting with miR-877-3p/PMEPA1 axis.

Abstract 1981: TGF-β induced PLEK2 promotes metastasis and chemoresistance in ESCC by up-regulation of LCN2

Abstract Background: Esophageal squamous cell carcinoma (ESCC) patients suffer from poor prognosis in general. Novel molecular markers to predict prognosis and serve as potential therapy target are much needed. Our previous research has established a comprehensive database of ESCC (GSE53625). Combined with data from TCGA, we found Pleckstrin 2 (PLEK2) predicts poor prognosis in ESCC. Meanwhile, its role in ESCC progression remains unknown. Methods: The TCGA database and GSE53625 are used to establish the expression profiles of PLEK2 and overall survival rates. Loss-of-function assays were performed to examine the effect of PLEK2 on proliferation, migration and invasion of ESCC cells in vitro and in vivo. ChIP assay was used to detect the relationship between TGF-β and PLEK2. Western blot and quantitative real time PCR (qRT-PCR) were carried out to reveal the interrelation between PLEK2 and LCN2. Results: PLEK2 had a higher expression in ESCCs and predicted poor overall survival (OS). PLEK2 attenuation by shRNA was sufficient to decrease ESCC cells proliferation, clonogenicity, migration and invasion in vitro and tumorigenicity and distant metastasis in vivo. Conversely, elevating PLEK2 levels performed opposite effects. Downregulation of PLEK2 led to higher proportions of apoptotic ESCC cells when induced by Cisplatin. The high expression of PLEK2 in ESCC patients also linked to shorter OS of patients who received adjuvant chemotherapy. TGF-β could increase the expression of PLEK2 by stimulation of Smad2/3 which can bind directly with the promoter sequences of PLEK2. Analyses of transcriptome profiling suggested LCN2 as the downstream of PLEK2. Overexpression LCN2 in PLEK2 stable knockdown ESCC cells can reverse the decreased migration and invasion, but not the proliferation of ESCC cells. TGF-β increased the expression of LCN2, but the effect disappeared when PLEK2 was knockdown. We also find that AKT pathway is involved in the whole regulatory processes. Conclusion: Our results revealed the significance of PLEK2 in driving oncogenesis and metastasis in ESCC by regulating LCN2 which activates AKT pathway. We also indicated that PLEK2 was associated with chemo-resistance. Our findings indicates the potential use of PLEK2 as a prognostic biomarker and a candidate therapeutic target in ESCC. Keywords: PLEK2; ESCC; TGF-β; chemo-resistance Citation Format: Feng Wang, Chaoqi Zhang, Chengming Liu, Hong Cheng, Nan Sun, Jie He. TGF-β induced PLEK2 promotes metastasis and chemoresistance in ESCC by up-regulation of LCN2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1981.

Activation of FXR Suppresses Esophageal Squamous Cell Carcinoma Through Antagonizing ERK1/2 Signaling Pathway.

IntroductionFarnesoid X receptor (FXR), a member of nuclear receptors, functionally regulates bile acid, glucose and lipid homeostasis. It is also worth noting that FXR plays a suppressor role in cancer and inflammation. However, the contribution of FXR to esophageal squamous cell carcinoma (ESCC) remains unknown.MethodsThe role of FXR activation in ESCC progression was evaluated in ESCC cell lines KYSE150 and EC109 in vitro and BALB-C nude mice in vivo. In vitro, FXR synthetic ligand GW4064 was used to detect the effects on ESCC cell proliferation, migration, apoptosis and cell cycles. To assess the effects of GW4064 on ESCC development in vivo, a xenograft tumor model was constructed. And ERK1/2 activity was evaluated by immunoblot analysis.ResultsFXR synthetic ligand GW4064 impaired esophageal squamous cell carcinoma (ESCC) proliferation and migration, induced apoptosis and cell cycle arrest in vitro, accompanied by inhibition of some inflammatory genes and promotion of pro-apoptotic genes. We then found that FXR activation decreased the phosphorylation levels of ERK1/2 induced by tumor necrosis factor-α (TNF-α) in ESCC cells. Consistent with these results, GW4064 suppressed ESCC tumorigenesis in a xenograft model and suppressed the phosphorylation of ERK1/2 in tumors.DiscussionThese findings identify that activating FXR may serve as a promising therapy or adjuvant therapeutic tool for controlling ESCC development.

Expression and Clinical Value of LncRNA GAPLINC in Esophageal Squamous Cell Carcinoma.

BackgroundThe long noncoding RNA (lncRNA) GAPLINC, or gastric adenocarcinoma predictive long intergenic ncRNA, plays a carcinogenic role in a variety of different tumor types. There is limited information regarding the biological function of GAPLINC in the development of esophageal squamous cell carcinoma (ESCC).MethodsSurgical tissue samples of 40 patients undergoing ESCC radical surgery were collected, including ESCC tissues and corresponding adjacent normal tissues. Quantitative real-time PCR (qRT-PCR) was used to detect the expression of lncRNA GAPLINC in the human ESCC cell line (TE11). The function role of LncRNA GAPLINC was detected after specific siRNA interference and overexpression in the TE11 cell line. The effects of LncRNA GAPLINC on ESCC cell proliferation, migration and invasion abilities were investigated by flow cytometry, using the Cell Counting Kit-8 (CCK-8), and by Transwell migration assays, respectively.ResultsThe expression of lncRNA GAPLINC in ESCC tissues was significantly higher than that in corresponding adjacent normal tissues (P<0.05) and correlated with the degree of tumor differentiation (P<0.05). Compared with human esophageal normal epithelial cell lines, the expression of LncRNA GAPLINC was significantly higher in the human ESCC cell line (P<0.05). CCK-8 assays showed that LncRNA GAPLINC overexpression increased the growth rate of cells (P<0.05). Transwell experiments showed that LncRNA GAPLINC overexpression increased the ability of cell migration and invasion compared to control cells (P<0.05). Annexin V assay revealed that LncRNA GAPLINC silencing increased early stage apoptosis (P<0. 05).ConclusionOur results suggest that LncRNA GAPLINC may be used as a biomarker for the diagnosis and monitoring of ESCC, and may play an oncogenic role in ESCC.

Circular RNA hsa_circ_0000277 sequesters miR-4766-5p to upregulate LAMA1 and promote esophageal carcinoma progression

Growing evidence has indicated that circular RNAs (circRNAs) play a pivotal role as functional RNAs in diverse cancers. However, most circRNAs involved in esophageal squamous cell carcinoma (ESCC) remain undefined, and the underlying molecular mechanisms mediated by circRNAs are largely unclear. Here, we screened human circRNA expression profiles in ESCC tissues and found significantly increased expression of hsa_circ_0000277 (termed circPDE3B) in ESCC tissues and cell lines compared to the normal controls. Moreover, higher circPDE3B expression in patients with ESCC was correlated with advanced tumor-node-metastasis (TNM) stage and dismal prognosis. Functional experiments demonstrated that circPDE3B promoted the tumorigenesis and metastasis of ESCC cells in vitro and in vivo. Mechanistically, bioinformatics analysis, a dual-luciferase reporter assay, and anti-AGO2 RNA immunoprecipitation showed that circPDE3B could act as a competing endogenous RNA (ceRNA) by harboring miR-4766-5p to eliminate the inhibitory effect on the target gene laminin α1 (LAMA1). In addition, LAMA1 was significantly upregulated in ESCC tissues and was positively associated with the aggressive oncogenic phenotype. More importantly, rescue experiments revealed that the oncogenic role of circPDE3B in ESCC is partly dependent on the miR-4766-5p/LAMA1 axis. Furthermore, bioinformatics analysis combined with validation experiments showed that epithelial-mesenchymal transition (EMT) activation was involved in the oncogenic functions of the circPDE3B–miR-4766-5p/LAMA1 axis in ESCC. Taken together, we demonstrate for the first time that the circPDE3B/miR-4766-5p/LAMA1 axis functions as an oncogenic factor in promoting ESCC cell proliferation, migration, and invasion by inducing EMT, implying its potential prognostic and therapeutic significance in ESCC.

LncRNA HCP5 promotes malignant cell behaviors in esophageal squamous cell carcinoma via the PI3K/AKT/mTOR signaling

ABSTRACT The role of lncRNA HCP5 in esophageal squamous cell carcinoma (ESCC) remains unknown despite its involvement in different malignancies. MTT assay, EdU assay, TUNEL assay, transwell assay, and sphere formation assay were conducted to reveal ESCC cell viability, proliferation, apoptosis, migration, invasion, and stemness characteristics. FISH and subcellular fraction assays were performed to reveal the subcellular location of HCP5 in ESCC cells. Luciferase reporter assay and RIP assay were conducted to explore the downstream axis of HCP5. Our findings revealed that HCP5 expression was at a higher level in ESCC tissues and cells compared to that in control tissues and cells. Additionally, HCP5 promoted ESCC cellular activities by promoting proliferation, migration, invasion ability and stemness characteristics of ESCC cells as well as suppressing cell apoptosis. Furthermore, we found that HCP5 bound with miR-139-5p to upregulate PDE4A via the competing endogenous RNA network in ESCC cells. Importantly, HCP5 was discovered to stimulate the PI3K/AKT/mTOR signaling by regulating the downstream target genes. Finally, rescue assays indicated that HCP5 promoted ESCC cell growth by activating the PDE4A-medaited PI3K/AKT/mTOR pathway. HCP5 promotes ESCC cellular development by modulating the miR-139-5p/PDE4A pathway and stimulating the PI3K/AKT/mTOR signaling pathway, which may be conducive for the improvement of ESCC treatment.

TRAIL promotes epithelial-to-mesenchymal transition by inducing PD-L1 expression in esophageal squamous cell carcinomas

BackgroundTumor necrosis factor-associated apoptosis-inducing ligand (TRAIL) was initially considered an immunity guard; however, its function remains controversial. Besides immune cells, lung and colon cancer cells have also been reported to express TRAIL, which can promote tumor invasion and metastasis. However, the biological function and underlying mechanism of action of TRAIL in esophageal squamous cell carcinoma (ESCC) remain poorly elucidated.MethodsThe ESCC cells stemness, migration, and proliferation ability was assessed by sphere formation, Transwell, and CCK8 assay. The stemness- and epithelial-mesenchymal transition (EMT)- related genes expression levels were analyzed by Western blot and RT-qPCR. The signal activation was conducted by Western blot. The xenograft mouse experiments and lung metastasis model were performed to confirm our findings in vitro.ResultsHerein, we found that TRAIL is a negative predictor in patients with ESCC. To further investigate the biological function of TRAIL, we established TRAIL knockdown and overexpression ESCC cell lines and found that TRAIL induced EMT and promoted tumor aggressiveness. Furthermore, we demonstrated that TRAIL- overexpressing cells upregulated PD-L1 expression, which was dependent on the p-ERK/STAT3 signaling pathway. We obtained similar results when using recombinant human TRAIL. Finally, we validated the biological role and mechanism of action of TRAIL in vivo.ConclusionsThese findings demonstrate that TRAIL promotes ESCC progression by enhancing PD-L1 expression, which induces EMT. This may explain the failure of TRAIL preclinical trials.

High Serum Elafin Prediction of Poor Prognosis of Locoregional Esophageal Squamous Cell Carcinoma

Simple SummaryConventional serum markers such as carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC), and tissue polypeptide antigen (TPA) have a low sensitivity in predicting the prognosis of locoregional esophageal squamous cell carcinoma cell (ESCC). In our clinical study, we found high serum elafin to be an independent outcome predictor for stage I-IIIA ESCC, considering T, N, overall stage, and treatment. In vitro experiments showed that adding recombinant elafin drove ESCC cell proliferation, migration and invasion, while shRNA attenuated elafin levels, abrogating those effects. Our results suggested serum elafin might be a noninvasive biomarker to predict the outcome of locoregional ESCC and could potentially be used as a therapeutic target.Esophageal squamous cell carcinoma (ESCC) is a highly aggressive tumor known to have locally advanced and metastatic features which cause a dismal prognosis. We sought to determine whether elafin, a non-invasive and secretory small-molecule marker, could be used to predict prognosis in locoregional ESCC patients in human and in vitro studies. In our human study, 119 subjects were identified as having incident and pathologically-proved ESCC with stage I-IIIA tumors from southern Taiwan between 2000 and 2016. We measured their serum elafin levels at baseline and followed them until the date of cancer death or until January 2020, the end of this study. Those with high serum elafin levels were found to have a 1.99-fold risk (95% confidence interval: 1.17–3.38) shorter survival than those who did not. In our in vitro experiments, elevated elafin levels were found to drive ESCC cell proliferation, migration and invasion, while attenuation of elafin level by shRNA abrogated those effects. We concluded that elafin promotes ESCC motility and invasion and leads to a worse clinical prognosis in ESCC patients without distant metastasis.

Overexpression of microRNA-19a-3p promotes lymph node metastasis of esophageal squamous cell carcinoma via the RAC1/CDC42-PAK1 pathway.

BackgroundA tendency towards extensive regional lymph node metastasis (LNM) is a typical clinical characteristic of esophageal squamous cell carcinoma (ESCC). Up-regulated microRNA (miR)-19a-3p was verified as a predictor of LNM in ESCC in previous microarray analyses, but the underlying mechanisms remain unclear. Here, in vitro experiments were performed to confirm the effect of miR-19a-3p on promoting LNM and to explore the underlying mechanisms.MethodsKYSE-150 and TE-1 cell lines were transfected with lentiviral vectors to inhibit miR-19a-3p (LV-miR-19a-3p-inhibition), and cell proliferation, invasion, and migration were assessed. Target genes of miR-19a-3p were identified by sequencing analysis and quantitative reverse transcription PCR (qRT-PCR); Western blotting was performed to confirm targets and explore the potential mechanisms underlying the effect of miR-19a-3p on LNM.ResultsmiR-19a-3p had no effect on ESCC cell proliferation, whereas miR-19a-3p overexpression promoted the invasion and migration of ESCC cells. qRT-PCR verification and western blot analysis showed that LV-miR-19a-3p-inhibition downregulated cell division cycle 42 (CDC42), Rac family small GTPase 1 (RAC1), and p21 activated kinase 1 (PAK1).ConclusionsOverexpression of miR-19a-3p increased the invasion and migration of ESCC cells via the RAC1/CDC42-PAK1 pathway, suggesting that this pathway mediates the effect of miR-19a-3p on promoting LNM in ESCC.

Aberrant MCM10 SUMOylation induces genomic instability mediated by a genetic variant associated with survival of esophageal squamous cell carcinoma.

BackgroundEsophageal squamous cell carcinoma (ESCC) is one of the common gastrointestinal malignancy with an inferior prognosis outcome. DNA replication licensing aberration induced by dysregulation of minichromosome maintenance proteins (MCMs) causes genomic instability and cancer metastasis. SUMOylation modification plays a pivotal role in regulation of genomic integrity, while its dysregulation fueled by preexisting germline variants in cancers remains poorly understood.MethodsFirstly, we conducted two‐stage survival analysis consisting of an exome‐wide association study in 904 ESCC samples and another independent 503 ESCC samples. Then, multipronged functional experiments were performed to illuminate the potential biological mechanisms underlying the promising variants, and MCM10 influences the ESCC progression. Finally, we tested the effects of MCM10 inhibitors on ESCC cells.ResultsA germline variant rs2274110 located at the exon 15 of MCM10 was identified to be significantly associated with the prognosis of ESCC patients. Individuals carrying rs2274110‐AA genotypes confer a poor survival (hazard ratio = 1.61, 95% confidence interval = 1.35–1.93, p = 1.35 × 10−7), compared with subjects carrying rs2274110‐AG/GG genotypes. Furthermore, we interestingly found that the variant can increase SUMOylation levels at K669 site (Lys[K]699Arg[R]) of MCM10 protein mediated by SUMO2/3 enzymes, which resulted in an aberrant overexpression of MCM10. Mechanistically, aberrant overexpression of MCM10 facilitated the proliferation and metastasis abilities of ESCC cells in vitro and in vivo by inducing DNA over‐replication and genomic instability, providing functional evidence to support our population finding that high expression of MCM10 is extensively presented in tumor tissues of ESCC and correlated with inferior survival outcomes of multiple cancer types, including ESCC. Finally, MCM10 inhibitors Suramin and its analogues were revealed to effectively block the metastasis of ESCC cells.ConclusionsThese findings not only demonstrate a potential biological mechanism between aberrant SUMOylation, genomic instability and cancer metastasis, but also provide a promising biomarker aiding in stratifying ESCC individuals with different prognosis, as well as a potential therapeutic target MCM10.