Chemoresistance In Esophageal Cancer Research Articles

Alarmin S100A8 imparts chemoresistance of esophageal cancer by reprogramming cancer-associated fibroblasts

Chemotherapy remains the first-line treatment for advanced esophageal cancer. However, durable benefits are achieved by only a limited subset of individuals due to the elusive chemoresistance. Here, we utilize patient-derived xenografts (PDXs) from esophageal squamous-cell carcinoma to investigate chemoresistance mechanisms in preclinical settings. We observe that activated cancer-associated fibroblasts (CAFs) are enriched in the tumor microenvironment of PDXs resistant to chemotherapy. Mechanistically, we reveal that cancer-cell-derived S100A8 triggers the intracellular RhoA-ROCK-MLC2-MRTF-A pathway by binding to the CD147 receptor of CAFs, inducing CAF polarization and leading to chemoresistance. Therapeutically, we demonstrate that blocking the S100A8-CD147 pathway can improve chemotherapy efficiency. Prognostically, we found the S100A8 levels in peripheral blood can serve as an indicator of chemotherapy responsiveness. Collectively, our study offers a comprehensive understanding of the molecular mechanisms underlying chemoresistance in esophageal cancer and highlights the potential value of S100A8 in the clinical management of esophageal cancer.

NGS-based profiling identifies miRNAs and pathways dysregulated in cisplatin-resistant esophageal cancer cells.

Esophageal cancer (EC) incidence remains to be on a global rise supported by an unchanged recurrence and 5-year survival rate owing to the development of chemoresistance. Resistance to cisplatin, one of the majorly used chemotherapeutic drugs in EC, is a major nuisance. This study sheds light on miRNA dysregulation and its inverse relation with dysregulated mRNAs to guide pathways into the manifestation of cisplatin resistance in EC. A cisplatin-resistant version of an EC cell line was established and comparative profiling by NGS with the parental cell line was employed to identify dysregulation in miRNA and mRNA levels. Protein-protein interaction network analysis was done using Cytoscape, followed by Funrich pathway analysis. Furthermore, selective significant miRNAs were validated using qRT-PCR. miRNA-mRNA integrated analysis was carried out using the Ingenuity Pathway Analysis (IPA) tool. Expression of various established resistance markers supported the successful establishment of cisplatin-resistant cell line. Whole-cell small RNA sequencing and transcriptome sequencing identified 261 miRNAs and 1892 genes to be significantly differentially expressed (DE), respectively. Pathway analysis indicated enrichment of EMT signaling, supported by NOTCH, mTOR, TNF receptor, and PI3K-mediated AKT signaling pathways, in chemoresistant cells. Validation by qRT-PCR confirmed upregulation of miR-10a-5p, miR-618, miR-99a-5p, and miR-935 and downregulation of miR-335-3p, miR-205-5p, miR-944, miR-130a-3p, and miR-429 in resistant cells. Pathway analysis that followed IPA analysis indicated that the dysregulation of these miRNAs and their target genes may be instrumental in the development and regulation of chemoresistance via p53 signaling, xenobiotic metabolism, and NRF2-mediated oxidative stress. This study concludes the interplay between miRNA and mRNA as an important aspect and occurrence in guiding the regulation, acquisition, and maintenance of chemoresistance in esophageal cancer in vitro.

Molecular mechanisms associated with chemoresistance in esophageal cancer.

Esophageal cancer (EC) is one of the most incident and lethal tumors worldwide. Although surgical resection is an important approach in EC treatment, late diagnosis, metastasis and recurrence after surgery have led to the management of adjuvant and neoadjuvant therapies over the past few decades. In this scenario, 5-fluorouracil (5-FU) and cisplatin (CISP), and more recently paclitaxel (PTX) and carboplatin (CBP), have been traditionally used in EC treatment. However, chemoresistance to these agents along EC therapeutic management represents the main obstacle to successfully treat this malignancy. In this sense, despite the fact that most of chemotherapy drugs were discovered several decades ago, in many cases, including EC, they still represent the most affordable and widely employed treatment approach for these tumors. Therefore, this review summarizes the main mechanisms through which the response to the most widely chemotherapeutic agents used in EC treatment is impaired, such as drug metabolism, apoptosis resistance, cancer stem cells (CSCs), cell cycle, autophagy, energetic metabolism deregulation, tumor microenvironment and epigenetic modifications.

Nicotine-Induced ILF2 Facilitates Nuclear mRNA Export of Pluripotency Factors to Promote Stemness and Chemoresistance in Human Esophageal Cancer.

Balancing mRNA nuclear export kinetics with its nuclear decay is critical for mRNA homeostasis control. How this equilibrium is aberrantly disrupted in esophageal cancer to acquire cancer stem cell properties remains unclear. Here we find that the RNA-binding protein interleukin enhancer binding factor 2 (ILF2) is robustly upregulated by nicotine, a major chemical component of tobacco smoke, via activation of JAK2/STAT3 signaling and significantly correlates with poor prognosis in heavy-smoking patients with esophageal cancer. ILF2 bound the THO complex protein THOC4 as a regulatory cofactor to induce selective interactions with pluripotency transcription factor mRNAs to promote their assembly into export-competent messenger ribonucleoprotein complexes. ILF2 facilitated nuclear mRNA export and inhibited hMTR4-mediated exosomal degradation to promote stabilization and expression of SOX2, NANOG, and SALL4, resulting in enhanced stemness and tumor-initiating capacity of esophageal cancer cells. Importantly, inducible depletion of ILF2 significantly increased the therapeutic efficiency of cisplatin and abrogated nicotine-induced chemoresistance in vitro and in vivo. These findings reveal a novel role of ILF2 in nuclear mRNA export and maintenance of cancer stem cells and open new avenues to overcome smoking-mediated chemoresistance in esophageal cancer. SIGNIFICANCE: This study defines a previously uncharacterized role of nicotine-regulated ILF2 in facilitating nuclear mRNA export to promote cancer stemness, suggesting a potential therapeutic strategy against nicotine-induced chemoresistance in esophageal cancer.

Mechanism of autophagy regulating chemoresistance in esophageal cancer cells

The current study aimed to explore the mechanism of autophagy-regulating chemoresistance in esophageal cancer (EC) cells. Methods: 45 cases of esophageal cancer cell tissue and 25 cases of adjacent normal tissue excised in the surgical resection were collected from the tumor pathology department of our hospital from March to November 2017. The above cancer cells and paracancerous cells were cultured according to the cell culture procedures. The autophagy was induced by cisplatin in human esophageal cancer EC9706 cells line. The effect of autophagy on the survival of EC9706 cells was observed by autophagy inhibitor 3-MA. Cell viability was also measured by cell counting kit-8 (CCK-8). Apoptosis and cell cycle were detected by flow cytometry. Furthermore, monodansylcadaverine (MDC) was used to detect autophagy. Western blot was applied to determine the molecular changes during treatment. Diketopyrrolopyrrole (DPP) is able to inhibit cell proliferation, induce cell death and cell cycle arrest in the S phase. In addition, autophagy was activated through PI3K-III pathway. Results: 3-MA inhibitor plus 10% fetal bovine serum were added for culture, and the cell culture temperature and humidity were the best conditions. There were few autophagic vesicles in the stationary cells, where their brightness was weakened. There were more and brighter green fluorescent particles in the DPP group without a 3-MA inhibitor, indicating that autophagic parameters actually exist in this process. The apoptosis rate of DDP-induced cell death was not found to be the best, but was higher than that of the control group (P<0.05). The combination of DDP and 3-MA had a more obvious catalytic effect on apoptosis, and the apoptosis rate was much higher than that of single DDP (P<0.05), indicating that DDP was capable of inducing significant apoptosis after inhibiting autophagy. The combination of DDP and 3-MA had an obvious catalytic effect on apoptosis, and the apoptosis rate was higher than that of DDP alone (P < 0.05), suggesting that DDP could significantly improve the ability to induce apoptosis after inhibiting autophagy. The expression level of autophagy-related proteins was also detected by Western blotting. Our findings indicated that autophagy may be a self-protective mechanism of esophageal cancer cells induced by DDP, and its inhibition may be a new strategy for adjuvant chemotherapy in esophageal cancer.

Targeting Wnt/β‐catenin by anthelmintic drug niclosamide overcomes paclitaxel resistance in esophageal cancer

Esophageal cancer is an aggressive malignancy, and its current treatment strategies are plagued with high rates of recurrence. In this work, we demonstrate that niclosamide, an anthelmintic drug, is a potential sensitizing candidate for overcoming chemoresistance in esophageal cancer. Using a panel of esophageal cancer cell lines and normal cells, we show that niclosamide has anti-esophageal cancer activity and is likely to be less effective against normal esophageal epithelial and fibroblast cells. The combination of niclosamide with paclitaxel results in much greater efficacy than paclitaxel alone, suggesting that niclosamide is active against esophageal cancer cells that are resistant to paclitaxel. This is further confirmed by our results that niclosamide is effective in inhibiting proliferation and inducing apoptosis in paclitaxel-resistant esophageal cancer cells. In line with the findings obtained from in vitro cell culture system, niclosamide augments the in vivo efficacy of paclitaxel and significantly arrests paclitaxel-resistant esophageal cancer growth without causing toxicity in mice. Mechanistically, we show that niclosamide decreases β-catenin level and activity, and inhibits phosphorylation of STAT3 and mTORC1 substrate 70S6K. Stabilization of β-catenin level by Wnt activator lithium chloride (LiCl) significantly abolishes the inhibitory effects of niclosamide in inhibiting proliferation and survival but not suppressing phosphorylation of STAT3 and 70S6K in paclitaxel-resistant esophageal cancer cells, suggesting that niclosamide sensitizes esophageal cancer cell to paclitaxel mainly through inhibiting Wnt/β-catenin. Our work demonstrates the efficacy of niclosamide and its underlying mechanism in paclitaxel-resistant esophageal cancer. Our work emphasizes that Wnt/β-catenin inhibition is a sensitizing strategy in esophageal cancer.

Tumor-infiltrating M2 macrophage in pretreatment biopsy sample predicts response to chemotherapy and survival in esophageal cancer.

The association between the tumor microenvironment (TME) and treatment response or survival has been a recent focus in several types of cancer. However, most study materials are resected specimens that were completely modified by prior chemotherapy; therefore, the unmodified host immune condition has not yet been clarified. The aim of the present study was to evaluate the relationship between TME assessed in pre–therapeutic biopsy samples and chemoresistance in esophageal cancer (EC). A total of 86 endoscopic biopsy samples from EC patients who received neoadjuvant chemotherapy (NAC) prior to surgery were evaluated for the number of intratumoral CD4+ lymphocytes (with/without Foxp3 expression), CD8+ lymphocytes (with/without PD‐1 expression), monocytes (CD14+) and macrophages (CD86+, CD163+ and CD206+) by multiplex immunohistochemistry (IHC). The number of tumor‐infiltrating CD206+ macrophages I significantly correlated with cT, cM, cStage and neutrophil/lymphocyte ratio (NLR), whereas the number of lymphocytes (including expression of Foxp3 and PD‐1) was not associated with clinico‐pathological features. The high infiltration of CD163+ or CD206+ macrophages was significantly associated with poor pathological response to NAC (P = 0.0057 and 0.0196, respectively). Expression of arginase‐1 in CD163+ macrophages tended to be higher in non–responders (29.4% vs 18.2%, P = 0.17). In addition, patients with high infiltration of M2 macrophages exhibited unfavorable overall survival compared to those without high infiltration of M2 macrophages (5‐year overall survival 57.2% vs 71.0%, P = 0.0498). Thus, a comprehensive analysis of TME using multiplex IHC revealed that M2 macrophage infiltration would be useful in predicting the response to NAC and long‐term survival in EC patients.

LncRNA CCAT1 is a biomarker for the proliferation and drug resistance of esophageal cancer via the miR-143/PLK1/BUBR1 axis.

Recent evidence indicates that long noncoding RNA colon cancer-associated transcript-1 (lncRNA CCAT1) is abundantly expressed in esophageal cancer and is closely related to the occurrence, development, invasion, metastasis, and drug resistance of this disease. However, the role and molecular mechanisms of CCAT1 in the cell proliferation and chemoresistance of esophageal cancer are largely unknown. The correlation between CCAT1 expression and drug resistance to cisplatin (CDDP) in esophageal squamous cell carcinoma (ESCC) cells was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and quantitative real-time polymerase chain reaction (qRT-PCR) assays. CCAT1 knockdown and miR-143 overexpression or inhibition were used to verify the effects on proliferation and drug resistance via MTT, western blotting, flow cytometry, and immunofluorescence assays. qRT-PCR and western blotting were applied to detect the potential regulatory relationship among CCAT1, miR-143, PLK1, and BUBR1. A xenograft tumor assay was performed to validate the role of CCAT1 in vivo. The expression of CCAT1 was positively correlated with drug resistance in several ESCC cell lines. CCAT1 knockdown and miR-143 overexpression inhibited cell proliferation and CDDP drug resistance. Moreover, the downstream target of CCAT1 was found to be miR-143, which can regulate the expression of PLK1 and BUBR1. In vivo assays showed that CCAT1 knockdown suppressed tumor growth and enhanced the sensitivity of tumors to CDDP in nude mice. Taken together, we discovered a novel mechanism by which CCAT1 promotes cell proliferation and enhances drug resistance by regulating the miR-143/PLK1/BUBR1 signaling axis both in vitro and in vivo. Our findings further suggest that lncRNA CCAT1 may be a potential therapeutic target for overcoming chemoresistance in esophageal cancer.

&lt;p&gt;CDKN3 promotes tumor progression and confers cisplatin resistance via RAD51 in esophageal cancer&lt;/p&gt;

Purpose: Esophageal cancer (ESCA) progression and chemoresistance are critical factors that impact the survival of patients with esophageal cancer. Cyclin dependent kinase inhibitor 3 (CDKN3) is an important regulator of the cell cycle that has received little attention, therefore the purpose of this study was to investigate CDKN3 involvement in ESCA.Methods: We first explored the public database in addition to our cohort to evaluate the expression of CDKN3 in ESCA patients. We performed bioinformative analysis on specific processes regulated by CDKN3, then we investigated the role of CDKN3 in ESCA progression and chemoresistance in vitro and in vivo. Finally, we sought to elucidate the mechanism of CDKN3 regulation of chemoresistance in ESCA.Results: We discovered that CDKN3 was highly expressed in ESCA and serves as an independent prognostic factor of this disease. Bioinformatic analysis showed CDKN3 involvement in DNA replication, the cell cycle G2/M phase transition, DNA damage repair (DDR) signaling pathways, et al Functional experiments in vitro and in vivo demonstrated that CDKN3 promoted ESCA progression and enhanced cisplatin resistance. Furthermore, CDKN3 inhibition resulted in reduced expression of RAD51, which plays a pivotal role in DDR. Overexpression of RAD51 reversed cisplatin-induced DNA damage and chemosensitivity in CDKN3 inhibited ESCA cell lines.Conclusion: The present research indicated that CDKN3 promoted ESCA progression and enhanced cisplatin resistance via RAD51, thereby influencing overall patient survival.

Identification of miR-29c and its Target FBXO31 as a Key Regulatory Mechanism in Esophageal Cancer Chemoresistance: Functional Validation and Clinical Significance.

Rationale: Dysregulated microRNA (miRNA) expressions in cancer can contribute to chemoresistance. This study aims to identify miRNAs that are associated with fluorouracil (5-FU) chemoresistance in esophageal squamous cell carcinoma (ESCC). The potential of miR-29c as a novel diagnostic, prognostic and treatment-predictive marker in ESCC, and its mechanisms and therapeutic implication in overcoming 5-FU chemoresistance were explored.Methods: The miRNA profiles of an ESCC cell model with acquired chemoresistance to 5-FU were analyzed using a Taqman miRNA microarray to identify novel miRNAs associated with 5-FU chemoresistance. Quantitative real-time PCR was used to determine miR-29c expression in tissue and serum samples of patients. Bioinformatics, gain- and loss-of-function experiments, and luciferase reporter assay were performed to validate F-box only protein 31 (FBXO31) as a direct target of miR-29c, and to identify potential transcription factor binding events that control miR-29c expression. The potential of systemic miR-29c oligonucleotide-based therapy in overcoming 5-FU chemoresistance was evaluated in tumor xenograft model.Results: MiR-29c, under the regulatory control of STAT5A, was frequently downregulated in tumor and serum samples of patients with ESCC, and the expression level was correlated with overall survival. Functional studies showed that miR-29c could override 5-FU chemoresistance in vitro and in vivo by directly interacting with the 3'UTR of FBXO31, leading to repression of FBXO31 expression and downstream activation of p38 MAPK. Systemically administered miR-29c dramatically improved response of 5-FU chemoresistant ESCC xenografts in vivo.Conclusions: MiR-29c modulates chemoresistance by interacting with FBXO31, and is a promising non-invasive biomarker and therapeutic target in ESCC.

Abstract 5125: The relationship between microbiome Fusobacterium nucleatum and autophagy in esophageal cancer

Abstract Background: Research into the microbiome is a rapidly advancing field in human cancers. The gut microbiome has recently been shown to play an important role in health, as well as in diseases such as obesity, inflammatory bowel disease, diabetes, non-alcoholic fatty liver disease, and several types of cancers. Fusobacterium nucleatum is a component of the human microbiome that primarily inhabits the oral cavity. It causes periodontal disease and has also been implicated in the development of human cancers. We have recently reported that Fusobacterium nucleatum in esophageal cancer tissues was associated with shorter survival, suggesting a potential role as a prognostic biomarker. In addition, It has been revealed that Fusobacterium nucleatum promotes chemoresistance to colorectal cancer by modulating autophagy. Nonetheless, the relationship between Fusobacterium nucleatum and autophagy in esophageal cancers have been still unknown. We constructed this study to examine the relationship between Fusobacterium nucleatum and autophagy. Methods: Using 284 esophageal cancers, we detected ATG-7 and ULK -1 expression by IHC staining in tumors. Results: 21% cases showed ATG-7 positive and 25% cases showed ULK-1 positive. ATG-7 and ULK-1 expression in Fusobacterium nucleatum-positive tumors were significantly higher compared with that of Fusobacterium nucleatum-negative tumors (P=0.026 and P=0.0077). In addition, ATG-7 expression was associated with cancer-specific survival rate in esophageal cancer patients (P=0.0496). There was no relationship of ULK-1 expression and prognosis. Conclusion: Fusobacterium nucleatum was related with ATG-7 and ULK-1. Our findings may shed lights on understanding the mechanism by which Fusobacterium nucleatum affects chemoresistance in esophageal cancer. Citation Format: Yang Liu, Yoshifumi Baba, Shigeki Nakagawa, Hiroshi Sawayama, Takatsugu Ishimoto, Masaaki Iwatsuki, Yoichi Yamashita, Yuji Miyamoto, Naoya Yoshida, Hideo Baba. The relationship between microbiome Fusobacterium nucleatum and autophagy 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 5125.

The Hippo Coactivator YAP1 Mediates EGFR Overexpression and Confers Chemoresistance in Esophageal Cancer.

Esophageal cancer is an aggressive malignancy and often resistant to therapy. Overexpression of EGFR has been associated with poor prognosis of patients with esophageal cancer. However, clinical trials using EGFR inhibitors have not provided benefit for patients with esophageal cancer. Failure of EGFR inhibition may be due to crosstalk with other oncogenic pathways. In this study, expression of YAP1 and EGFR were examined in EAC-resistant tumor tissues versus sensitive tissues by IHC. Western blot analysis, immunofluorescence, real-time PCR, promoter analysis, site-directed mutagenesis, and in vitro and in vivo functional assays were performed to elucidate the YAP1-mediated EGFR expression and transcription and the relationship with chemoresistance in esophageal cancer. We demonstrate that Hippo pathway coactivator YAP1 can induce EGFR expression and transcription in multiple cell systems. Both YAP1 and EGFR are overexpressed in resistant esophageal cancer tissues compared with sensitive esophageal cancer tissues. Furthermore, we found that YAP1 increases EGFR expression at the level of transcription requiring an intact TEAD-binding site in the EGFR promoter. Most importantly, exogenous induction of YAP1 induces resistance to 5-fluorouracil and docetaxcel, whereas knockdown of YAP1 sensitizes esophageal cancer cells to these cytotoxics. Verteporfin, a YAP1 inhibitor, effectively inhibits both YAP1 and EGFR expression and sensitizes cells to cytotoxics. Our data provide evidence that YAP1 upregulation of EGFR plays an important role in conferring therapy resistance in esophageal cancer cells. Targeting YAP1-EGFR axis may be more efficacious than targeting EGFR alone in esophageal cancer.

MiR-27 is associated with chemoresistance in esophageal cancer through transformation of normal fibroblasts to cancer-associated fibroblasts.

There is increasing evidence that the expression of microRNA (miRNA) in cancer is associated with chemosensitivity but the mechanism of miRNA-induced chemoresistance has not been fully elucidated. The aim of this study was to examine the role of extracellular miRNA in the response to chemotherapy in esophageal cancer. First, serum expression of miRNAs selected by miRNA array was measured by quantitative reverse transcription-polymerase chain reaction in 68 patients with esophageal cancer who received cisplatin-based chemotherapy to examine the relationship between miRNA expression and response to chemotherapy. The serum expression levels of 18 miRNAs were different between responders and non-responders by miRNA array. Of these, high expression levels of miR-27a/b correlated with poor response to chemotherapy in patients with esophageal cancer. Next, in vitro assays were conducted to investigate the mechanism of miRNA-induced chemoresistance. Although transfection of miR-27a/b to cancer cells had no significant impact on chemosensitivity, esophageal cancer cells cultured in supernatant of miR-27a/b-transfected normal fibroblast showed reduced chemosensitivity to cisplatin, compared with cancer cells cultured in supernatant of normal fibroblast. MiR-27a/b-transfected normal fibroblast showed α-smooth muscle actin (α-SMA) expression, a marker of cancer-associated fibroblasts (CAF) and increased production of transforming growth factor-β (TGF-β). Chemosensitivity recovered after administration of neutralizing antibody of TGF-β to the supernatant transfer experiments. Our results indicated that miR-27a/b is involved in resistance to chemotherapy in esophageal cancer, through miR-27a/b-induced transformation of normal fibroblast into CAF.