The lncRNA PARylator promotes PARP1 activation and resistance to DNA‑damaging therapy in esophageal squamous cell carcinoma

Interaction between Esophageal Squamous Cell Carcinoma and Adipose Tissue in Vitro

Drug resistance restrains the efficacy of platinum-based chemotherapy against esophageal squamous cell carcinoma (ESCC). Long noncoding RNAs (lncRNAs) play crucial roles in various cancers. In this study, we conducted bioinformatical analysis to search for key lncRNAs involved in ESCC progression. Experimental validations focused on a candidate lncRNA, HMGA2-AS1. The roles of the lncRNA HMGA2-AS1 in ESCC growth and cisplatin resistance were explored. The clinical significance of HMGA2-AS1 in ESCC was also determined. Our data demonstrated that HMGA2-AS1 was significantly upregulated in ESCC relative to adjacent normal tissues and correlated with tumor size (p = 0.0035) and tumor stage (p = 0.0001). Knockdown of HMGA2-AS1 suppressed the proliferation and colony formation and increased cisplatin sensitivity in ESCC cells. In vivo tumorigenic studies showed that depletion of HMGA2-AS1 impaired tumorigenesis of ESCC cells. Enforced expression of HMGA2-AS1 promoted ESCC proliferation and cisplatin resistance. HMGA2-AS1 interacted with HMGA2 protein in the nucleus of ESCC cells, promoting HMGA2-dependent transactivation of Snail2. Silencing of HMGA2 abrogated HMGA2-AS1-induced proliferation and cisplatin resistance in ESCC cells, which were rescued by overexpression of Snail2. In conclusion, our results highlight the importance of HMGA2-AS1 in ESCC growth and cisplatin resistance. HMGA2-AS1-induced aggressive phenotype in ESCC depends on the interaction with HMGA2. HMGA2-AS1 may serve as a potential therapeutic target for ESCC.

miR-516b functions as a tumor suppressor by directly modulating CCNG1 expression in esophageal squamous cell carcinoma

To evaluate the regulatory effects of lncRNA LINC00261 on proliferation, invasion, and metastasis of esophageal squamous cell carcinoma (ESCC) cells. The differentially expressed RNAs in ESCC were identified using the GSE149612 dataset from the GEO database. PCR was used to detect LINC00261 expression levels in clinical ESCC and normal esophageal tissue samples and in multiple ESCC cell lines and normal esophageal epithelial cells (HEEC). In ESCC cells, the effects of overexpression of LINC00261 on cell proliferation, invasion, metastasis and apoptosis were analyzed using CCK-8 assay, clone formation assay, Transwell assay and flow cytometry. The potential targets of LINC00261 were predicted using bioinformatics tools including ENCORI and verified using dual-luciferase reporter assay and Western blotting. The effects of LINC00261 overexpression on ESCC were confirmed in a nude mouse model bearing ESCC xenograft. Analysis of the GSE149612 dataset revealed significantly lower LINC00261 expression in ESCC tissues and cell lines. In cultured ESCC cells, LINC00261 overexpression markedly suppressed cell proliferation, invasion, and metastasis and promoted cell apoptosis. Dual-luciferase reporter assays confirmed that LINC00261 targets the miR-23a-3p/ZNF292 axis. In the tumor-bearing mouse model, LINC00261 overexpression significantly inhibited ESCC xenograft proliferation and metastasis. LINC00261 suppresses ESCC progression by targeting the miR-23a-3p/ZNF292 axis, suggesting a potential therapeutic strategy for ESCC treatment.

Introduction: Notch signaling may act as a tumor suppressor during the development of squamous cell carcinomas (SCCs); yet, Notch activation promotes tumor growth in a subset of SCC cells. The roles of Notch in the pathogenesis of esophageal squamous cell carcinoma (ESCC) remain elusive. Methods: Notch1 activation and epithelial-mesenchymal transition (EMT) were determined in an esophageal epithelium-targeted cell-lineage traceable (K5CreERT2-Rosa26tdTomatolsl) mouse model of ESCC induced by 4-nitroquinoline 1-oxide (4NQO), which was coupled with flow cytometry and single cell-derived ESCC organoid formation assays. Tumor-initiating capability was assessed in xenograft transplantation experiments with TE11 human ESCC cells carrying either Crispr-Cas9-deleted Notch1 loci or tetracycline-inducible expression of the activated form of Notch1 (ICN1). Surgically resected primary tumors and adjacent normal mucosa from ESCC patients (n = 152) were analyzed by immunohistochemistry for Notch1 activation and the EMT marker ZEB1. Results: 4NQO-treated mice developed tdTomato-positive primary and metastatic ESCC tumors with EpCAM-negative ESCC cells displaying traits compatible with EMT. Notch1 activation and ZEB1 expression were co-localized in ESCC cells at the stromal interface, a finding that was further recapitulated in ESCC tumor organoids. Interestingly, Cre-mediated ex vivo Notch1 deletion in a single cell suspension from Notch1loxP/loxP mouse-derived ESCC tumors decreased organoid formation rate. TE11 xenograft tumors appeared to contain a unique ESCC cell fraction containing EpCAM-negative cells, where ICN1 conferred tumorigenicity upon serial transplantation. This population displayed upregulation of Notch1 target genes and the ESCC-lineage survival factor/oncogene SOX2. Moreover, Notch1 deletion in TE11 not only limited tumor formation, but also decreased EMT in culture. A subset of ESCC patients (49/140, 33%) showed ICN1-positive ESCC cells with concurrent ZEB1 expression at the tumor invasive front. The presence of such ESCC cells was associated with poor 5-year survival (P = 0.001), tumor depth (P = 0.01), lymphatic and venous invasion (P = 0.003) and distant metastasis (P = 0.002). Moreover, such ICN1-expressing cells were increased in ESCC patients (7/12, 58%) who received pre-surgical neoadjuvant therapy. Conclusions: Cell-lineage tracing experiments validate for the first time Notch1 activation and EMT in the 4NQO-induced mouse model of ESCC. Analyses of single cell-derived ESCC tumor organoids, xenograft and primary ESCC tumors reveal that Notch1 activation may be associated with tumor initiating capability, EMT and chemotherapy resistance, implicating Notch1 activation in the pathogenesis of ESCC and potentially other SCCs. Citation Format: Koji Tanaka, Kelly A. Whelan, Naryan L. Rustgi, Prasanna M. Chandramouleeswaran, Seiji Naganuma, Shingo Kagawa, Mitsuteru Natsuizaka, Yoshiaki Kita, Shoji Natsugoe, Que Jianwen, Devraj Basu, Andres J. Klein-Szanto, Adam Bass, J. Alan Diehl, Hiroshi Nakagawa. Notch1 regulates epithelial-mesenchymal transition and tumor-initiating capability in esophageal squamous-cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2678.

The nuclear receptor Nur77 plays paradoxical roles in numerous cancers. However, whether Nur77 inhibits esophageal squamous cell carcinoma (ESCC) growth and affects immunological responses against ESCC has not been determined. The functional role of Nur77 in ESCC was investigated in this study using human ESCC cell lines, quantitative real-time polymerase chain reaction (PCR), cell proliferation and colony formation assays, flow cytometry analysis, western blotting and animal models. The target gene controlled by Nur77 was verified using dual-luciferase reporter assays, chromatin immunoprecipitation analysis and functional rescue experiments. To examine the clinical importance of Nur77, 72 human primary ESCC tissues were subjected to immunohistochemistry. Taken together, these findings showed that, both in vitro and in vivo, Nur77 dramatically reduced ESCC cell growth and triggered apoptosis. Nur77 directly interacts with the interferon regulatory factor 1 (IRF1) promoter to inhibit its activity in ESCC. Pharmacological induction of Nur77 using cytosporone B (CsnB) inhibited ESCC cell proliferation and promoted apoptosis both in vitro and in vivo. Furthermore, CsnB increased CD8+ T-cell infiltration and cytotoxicity to inhibit the formation of ESCC tumors in an immunocompetent mouse model. In ESCC tissues, Nur77 expression was downregulated, and IRF1 expression was increased; moreover, their expression levels were negatively related. IRF1 and Nur77 were strongly correlated with overall survival. These findings suggested that Nur77 targets and regulates the IRF1/PD-L1 axis to serve as a tumor suppressor in ESCC.Graphical abstract of the regulatory mechanism of Nur77 overexpression downregulates IRF1 in the inhibition of ESCC progression and enhance anti-PD-1 therapy efficacy.

Polygalacin D suppresses esophageal squamous cell carcinoma growth and metastasis through regulating miR-142-5p/Nrf2 axis

Objective: To investigate the role of microRNA-139-5p (miR-139-5p) in the occurrence and development of esophageal squamous cell carcinoma (ESCC) and its effects on cell proliferation and invasion of ESCC cells and its molecular mechanisms. Methods: Seventy-five cases of ESCC tissues and paired normal tissues were obtained from thoracic surgery of the First Affiliated Hospital of Zhengzhou University from February 2017 to March 2018. Experiment was divided into two group: ESCC (n=75) and normal esophageal tissues (n=75).GEO datasets and real-time quantitative PCR (qRT-PCR) were used to detect the expression of miR-139-5p in ESCC tissues and cells. miR-139-5p inhibitor, miR-139-5p mimic, negative control, control siRNA, T-box transcliption factor 1(TBX1) siRNA, pcDNA3.1 and pcDNA3.1-TBX1 were transfected into ESCC Eca109 and TE1 cells. qRT-PCR was used to detect the expressions of miR-139-5p and TBX1 in transfected ESCC cells. Cell counting kit 8 (CCK-8) and Transwell chamber were employed to detect cell proliferation and invasion of ESCC cells, respectively. Dual-Luciferase Reporter assay was used to analyze the interaction between miR-139-5p with TBX1. qRT-PCR, Western blot and immunohistochemistry were utilized to detect the expression of TBX1 in ESCC tissues. Western blot was used to detect the expressions of E-cadherin, N-cadherin and Vimentin after transfection. Results: The level of miR-139-5p in ESCC tissues was significantly lower than that in normal tissues (1.17±0.43 vs 5.16±3.62,P<0.001). Log-rank test showed that the survival rate of ESCC patients with high miR-139-5p level (n = 43) was significantly higher than that with low miR-139-5p level (n=32) (67.44% vs 25.00%, P = 0.005). The expression level of miR-139-5p in ESCC cells was significantly lower than that of normal esophageal epithelial cell Het-1A (all P<0.001). The proliferation and invasion ability of ECA109 and TE1 cells with high expression of miR-139-5p were significantly lower than those transfected with negative control (NC) (all P<0.05). Dual-Luciferase Reporter assay showed that miR-139-5p could bind to the 3'-untranslated region of TBX1. miR-139-5p mimic or inhibitor suppressed or promoted the expression of TBX1 protein in Eca109 and TE1 cells, respectively (all P<0.05). Downregulation of TBX1 significantly suppressed proliferation and invasion of ECA109 and TE1 cells, while overexpression of TBX1 significantly promoted proliferation and invasion of ECA109 and TE1 cells (all P<0.05). In addition, pcDNA3.1-TBX1 partially reversed the inhibition of miR-139-5p-mediated invasion ability (all P<0.05), while TBX1 siRNA partially reversed the enhancement of miR-139-5p inhibitor-mediated invasion ability (all P<0.05). Conclusion: miR-139-5p suppressed ESCC cell proliferation and invasion by targeting TBX1.

BackgroundCis-diamminedichloro-platinum (CDDP)-based chemotherapy regimens are the most predominant treatment strategies for patients with esophageal squamous cell carcinoma (ESCC). Dysregulated long non-coding RNAs (lncRNAs) contribute to CDDP resistance, which results in treatment failure in ESCC patients. However, the majority of lncRNAs involved in CDDP resistance in ESCC remain to be elucidated.MethodsThe public Gene Expression Omnibus (GEO) dataset GSE45670 was analysed to reveal potential lncRNAs involved in CDDP resistance of ESCC. Candidate upregulated lncRNAs were detected in ESCC specimens by qRT-PCR to identify crucial lncRNAs. Non-coding RNA activated by DNA damage (NORAD) was selected for further study. Kaplan-Meier analysis and a COX proportional regression model were performed to analyse the potential of NORAD for predicting prognosis of ESCC patients. The role of NORAD in CDDP resistance were determined by conducting gain and loss-of-function experiments in vitro. Fluorescence in situ hybridization (FISH) was performed to determine the subcellular location of NORAD in ESCC cells. A public GEO dataset and bioinformatic algorithms were used to predict the microRNAs (miRNAs) that might be latently sponged by NORAD. qRT-PCR was conducted to verify the expression of candidate miRNAs. Luciferase reporter and Argonaute-2 (Ago2)-RNA immunoprecipitation (RIP) assays were conducted to evaluate the interaction between NORAD and candidate miRNAs. A miRNA rescue experiment was performed to authenticate the NORAD regulatory axis and its effects on CDDP resistance in ESCC cells. Western blotting was conducted to confirm the precise downstream signalling pathway of NORAD. A xenograft mouse model was established to reveal the effect of NORAD on CDDP resistance in vivo.ResultsThe expression of NORAD was higher in CDDP-resistant ESCC tissues and cells than in CDDP-sensitive tissues and cells. NORAD expression was negatively correlated with the postoperative prognosis of ESCC patients who underwent CDDP-based chemotherapy. NORAD knockdown partially arrested CDDP resistance of ESCC cells. FISH showed that NORAD was located in the cytoplasm in ESCC cells. Furthermore, overlapping results from bioinformatic algorithms analyses and qRT-PCR showed that NORAD could sponge miR-224-3p in ESCC cells. Ago2-RIP demonstrated that NORAD and miR-224-3p occupied the same Ago2 to form an RNA-induced silencing complex (RISC) and subsequently regulated the expression of metadherin (MTDH) in ESCC cells. The NORAD/miR-224-3p/MTDH axis promoted CDDP resistance and progression in ESCC cells by promoting nuclear accumulation of β-catenin in vitro and in vivo.ConclusionsNORAD upregulates MTDH to promote CDDP resistance and progression in ESCC by sponging miR-224-3p. Our results highlight the potential of NORAD as a therapeutic target in ESCC patients receiving CDDP-based chemotherapy.

BackgroundAnoikis resistance has been demonstrated to facilitate distant metastases of cancers. MicroRNA-133b (miR-133b) is found to be down-regulated in various tumors, including esophageal squamous cell carcinoma (ESCC), and closely correlates with the malignant phenotype of ESCC. This study aimed to evaluate the roles of miR-133b in metastases of ESCC via regulating anoikis.MethodsThe expression of miR-133b and related molecules were detected in ESCC tissues and cells. The target relationship between miR-133b and epidermal growth factor receptor (EGFR) was verified by dual luciferase reporter assay. Cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Anoikis and anchorage-independent growth were assessed by anoikis assay and soft agar assay. Migration and invasion were evaluated by scratch and transwell assays. The expressions of related molecules were detected by reverse transcription-quantitative polymerase chain reaction and western blotting. The in vivo results were determined by tumor xenografts in nude mice.ResultsMiR-133b level was decreased in ESCC tissues and cells, which negatively correlated with EGFR, integrin β4 (ITGB4), and phosphorylated focal adhesion kinase levels. Moreover, miR-133b down-regulated EGFR expression in ESCC cells. Overexpression of miR-133b inhibited the anoikis resistance, migration, invasion and epithelial-mesenchymal transition of ESCC cells via targeting EGFR. Finally, miR-133b overexpression suppressed tumor growth and lung metastases of ESCC in vivo. ITGB4/FAK/growth factor receptor-bound protein 2 (Grb2), protein kinase B (AKT), and extracellular signal-regulated kinase (ERK) pathways were involved in the regulatory mechanisms of miR-133b/EGFR axis in ESCC metastases in vitro and in vivo.ConclusionsThe results suggested that miR-133b/EGFR axis regulated metastases of ESCC by affecting anoikis resistance via ITGB4/FAK/Grb2, AKT, and ERK pathways.

It is well known that garlics contain a large number of organosulfur compounds including diallyl trisulfide (DATS), which possess anticancer properties. However, the effects of DATS on esophageal squamous cell carcinoma (ESCC) growth are still poorly understood. In this study, we investigated the effects of DATS on ESCC cell growth in vivo and in vitro, as well as the associated signaling pathways. Cell proliferation was measured using the crystal violet assay. The transwell method was used to evaluate the effect of DATS on ESCC cell migration. Also, Western blot was performed to detect the activation of ERK1/2 and AKT1 responds to DATS. Finally, the effect of DATS on ESCC xenografts in nude mice was also investigated. Our results showed that DATS significantly inhibited ESCC cell proliferation in a time- and dose-dependent manner. DATS time-dependently (p < 0.05) increased phosphorylation of ERK1/2, but not AKT1. Suppression of ERK1/2 activation with PD9805 also completely blocked DATS-inhibited ESCC cell proliferation. Meanwhile, DATS also robustly suppressed ESCC xenograft growth and increased ERK1/2 activation in nude mice. Our finding demonstrated that DATS inhibits the proliferation of ESCC cells by activation of ERK1/2 in vitro and in vivo. These findings revealed that DATS could be used for therapeutic intervention for human ESCC.

Metastasis is one of most lethal causes that confer a poor prognosis of patients with esophageal squamous cell carcinoma (ESCC), whereas there is no available target drug for metastatic ESCC currently. In this study, we aimed to determine whether the transcriptional inhibition by CDK7/9 inhibitor SNS-032 is activity against ESCC. MTT and soft agar assays were performed to examine the influence of SNS-032 on ESCC growth in vitro. Tumor xenograft in nude mice was used to assess the antitumor activity of SNS-032 in vivo. The roles of SNS-032 in ESCC metastasis were conducted by wound healing and transwell assays in vitro, and by a lung and a popliteal lymph node metastasis model in vivo. The results showed that CDK7 and CDK9 were highly expressed in ESCC cells; SNS-032 effectively inhibited cellular viability, abrogated anchorage-independent growth, and potentiated the sensitivity to cisplatin in ESCC cells in vitro and in vivo. In addition, SNS-032 induced a mitochondrial-dependent apoptosis of ESCC cells by reducing Mcl-1 transcription. SNS-032 also potently abrogated the abilities of ESCC cell migration and invasion through transcriptional downregulation of MMP-1. Importantly, SNS-032 remarkably inhibited the growth of ESCC xenograft, increased the overall survival, as well as diminished the lung and lymph node metastasis in nude mice. Taken together, our findings highlight that the CDK7/9 inhibitor SNS-032 is a promising therapeutic agent, and warrants a clinical trial for its efficacy in ESCC patients, even those with metastasis.

Esophageal squamous cell carcinoma (ESCC) is common cancer in China. At the same time, microRNA-196b (miR-196b) has different promotion/inhibition effects in different cancers. The study aims to reveal the role of miR-196b in ESCC and explore its prognostic value. The expression of miR-196b in ESCC samples and cell lines was detected to explore the expression pattern of miR-196b in ESCC. Kaplan-Meier method was conducted for survival rate and Multivariate Cox analysis was used to explore the clinical significance of miR-196b in ESCC. The Cell Counting Kit-8 (CCK-8) assay, transwell migration and invasion tests were used to determine the biological function of miR-196b in ESCC. The relationship of miR-196b and SOCS2 in ESCC was detected by luciferase activity assay and RIP assay. Both in ESCC tissues and cell lines, miR-196b expression was up-regulated. miR-196b expression is related to TNM stage and lymph node metastasis. Combining with the results of Multivariate Cox regression analysis, miR-196b may be a potential independent prognostic marker for ESCC patients. The results of the functional analysis showed that miR-196b inhibitor can reduce cell proliferation, migration and invasion in ESCC cells. Besides, the suppressor of cytokine signaling 2 (SOCS2) is the target of miR-196b in ESCC. miR-196b may exist as a tumor-promoting factor in ESCC and enhance the proliferation abilities, migration capacities, and invasion potential of ESCC cells by targeting SOCS2. miR-196b and SOCS2 have a close negative correlation in ESCC, which may be used as a clinically poor prognostic biomarker and therapeutic target for ESCC.

The aim of this study was to explore the expression of long non-coding ribonucleic acid (lncRNA) nuclear receptor subfamily 2 group F member 1-antisense RNA 1 (NR2F1-AS1) in esophageal squamous cell carcinoma (ESCC) tissues and cells and to investigate its effects on ESCC proliferation and metastasis. The expression level of NR2F1-AS1 in 51 pairs of ESCC tissues and corresponding adjacent tissues was detected via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Meanwhile, NR2F1-AS1 expression in ESCC cells was measured via qRT-PCR as well. Subsequently, specific interference sequences of NR2F1-AS1 were designed, synthesized, and transiently transfected into ESCC cells. 48 h later, qRT-PCR assay was performed to detect the interference efficiency. The effects of small interfering (si)-NR2F1-AS1 on the proliferation of ESCC cells were determined through cell counting kit-8 (CCK-8) and colony formation assay. Wound healing and transwell assays were conducted to investigate the influences of si-NR2F1-AS1 on the migration and invasion of ESCC cells. Additionally, the changes in the expressions of epithelial-mesenchymal transition (EMT) molecular markers were detected by Western blotting. QRT-PCR assay revealed that the expression level of NR2F1-AS1 was significantly up-regulated in 42 of 51 cases of ESCC tissues (42/51, 82.4%). Compared with esophageal mucosal epithelial HET-1A cells, NR2FA-AS1 was highly expressed in ESCC cells. CCK-8 and colony formation assay indicated that the proliferation of ESCC cells decreased remarkably after interference in NR2F1-AS1 expression. The results of wound healing and transwell assays showed that the migration and metastasis of cells were significantly lower in si-NR2F1-AS1 group than those in si-NC group. Western blotting demonstrated that the expressions of EMT molecular markers were changed after interfering with NR2F1-AS1 expression. NR2F1-AS1 was highly expressed in ESCC tissues and cells. Furthermore, high expression of NR2F1-AS1 promoted the proliferation and metastasis of ESCC cells by modulating EMT.

Desmoplasia, or deposition of connective tissue proper in the stroma, is a key feature of esophageal squamous cell carcinoma (ESCC), a common malignancy worldwide. The cellular component of connective tissue proper is comprised of fibroblasts, and cancer-associated fibroblasts (CAFs) are widely recognized as a major constituent of the tumor microenvironment (TME). The purpose of this study was to investigate the cross-talk between CAFs and tumor cells in ESCC, as well as identify factors that mediate this cross-talk and evaluate these factors as therapeutic targets in ESCC and potentially other squamous cell carcinomas. We have observed that co-culture with CAFs prompts ESCC cells to acquire a more mesenchymal phenotype and express potent mediators of cell migration and invasion, such as matricellular proteins (periostin, fibrillin, osteonectin) and growth factors (EGF, HGF, VEGF). Furthermore, co-culture of ESCC cells with CAFs promotes tumor cell migration in our 3D organotypic culture, while co-transplantation of ESCC cells with fibroblasts leads to enhanced tumor growth in vivo, compared to tumors transplanted without fibroblasts. In order to identify potential mediators of ESCC-CAF cross-talk, we have performed a comprehensive cytokine array and found that interleukin 6 (IL-6) is significantly overexpressed in conditioned media from co-culture of esophageal CAFs and ESCC cells, compared to mono-culture. IL-6 is known to play important roles in the development of multiple types of cancer, mostly via activation of the STAT3 signaling pathway. To confirm relevance of our in vitro findings, we have performed immunohistochemical staining of tissue samples from ESCC patients and found that, compared to normal esophagus, expression of IL-6 is enhanced in ESCC in both epithelial cells and fibroblasts. Interestingly, knockdown of IL-6 resulted in altered morphology of ESCC cells in 3D organotypic culture. This was accompanied by restored membrane localization of E-cadherin and reduced activation of STAT3 signaling. In order to investigate the importance of STAT3 and MEK/ERK signaling (the latter being another pathway activated by IL-6 in cancer) on ESCC cell biology, we have treated 3D organotypic cultures with stattic and trametinib (inhibitors of STAT3 and MEK, respectively). This treatment resulted in impaired invasion, reduced proliferation and enhanced apoptosis of ESCC cells, accompanied by decreased expression of the basal cell marker p63. Furthermore, treatment with stattic and trametinib leads to reduced secretion of IL-6 by these cultures. These effects were more pronounced upon treatment with combination of stattic and trametinib. Since we found IL-6 to be important for ESCC progression in vitro, we have conducted a therapeutic study utilizing tocilizumab (an FDA-approved anti-human IL-6R neutralizing antibody). Subcutaneous xenograft tumors comprised of human ESCC and CAF cell lines, were treated with tocilizumab, which resulted in suppressed tumor growth, accompanied by decreased activation of STAT3 and ERK within the tumor, compared to control IgG-treated tumors. Furthermore, tocilizumab-treated animals had reduced numbers of splenic monocytic immature myeloid cells (CD11b+Ly-6C+), which is in agreement with previously published findings regarding MDSCs in murine models of ESCC. In summary, our findings indicate that IL-6, at least in part, is responsible for migration, proliferation, survival and poor differentiation of ESCC cells, promoted by CAFs in the TME. We also report that these effects of IL-6 are mediated by STAT3 and ERK signaling. Finally, we demonstrate that inhibition of IL-6 signaling suppresses ESCC tumor growth in vivo. This is the first report of anti-tumorigenic activity of tocilizumab in ESCC, which makes it a promising candidate for ESCC therapy. Supported by NCI P01 CA098101. Citation Format: Tatiana A. Karakasheva, Monica Soni, Todd J. Waldron, Eric W. Lin, Philip D. Hicks, Andres Klein-Szanto, Kwok K. Wong, Adam J. Bass, Anil K. Rustgi. Il-6 initiates a self-sustaining signaling loop to promote nurturing tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A32.