Ohara Toshiaki (Orcid ID: 0000-0003-1557-1589) Noma Kazuhiro (Orcid ID: 0000-0002-5584-3908) Tazawa Hiroshi (Orcid ID: 0000-0003-4658-1050) Fujiwara Toshiyoshi (Orcid ID: 0000-0002-5377-6051)
Research article
Nanog is a promising chemo-resistant stemness marker and therapeutic target by iron chelators for esophageal cancer
Short title: Nanog is a chemo-resistant stemness marker
Toru Narusaka 1, Toshiaki Ohara 1,2*, Kazuhiro Noma 1, Noriyuki Nishiwaki 1, Yuki Katsura 1, Takuya Kato 1, Hiroaki Sato 1, Yasuko Tomono 3, Satoru Kikuchi 1, Hiroshi Tazawa 1,4, Yasuhiro Shirakawa 1,5, Akihiro Matsukawa 2 and Toshiyoshi Fujiwara 1
1 Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
2 Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
3 Shigei Medical Research Institute, Okayama, Japan
4 Center for Innovative Clinical Medicine, Okayama University Hospital, Okayama, Japan
5 Department of Surgery, Hiroshima City Hiroshima Citizens Hospital, Hiroshima, Japan
*Correspondence: Toshiaki Ohara
Department of Pathology and Experimental Medicine
Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan
Tel: +81-86-235-7143; Fax: +81-86-235-7148 E-mail: [email protected]
Abstract: Esophageal cancer is a disease showing poor prognosis. Although combination chemotherapy using cisplatin and 5-fluorouracil is standard for unresectable esophageal cancer, the response rate is 35%. Cancer stem cells (CSCs) and inflammation are reportedly responsible for the poor prognosis of esophageal cancer. However, comprehensive analyses have not been conducted and proposals for progress remain lacking. Iron is known to be a key factor in the stemness of CSCs.
This study focused on the therapeutic potential of iron control using iron chelators for CSCs in esophageal cancer. Among 134 immunohistochemically analyzed cases, Nanog expression was high in 98 cases and low in 36 cases. High Nanog expression correlated with low overall and disease-free This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/ijc.33544
This article is protected by copyright. All rights reserved.
survivals. The iron chelators deferasirox (DFX) and SP10 suppressed the proliferation and expression of stemness markers in TE8 and OE33 cells. DFX and SP10 did not induce compensatory interleukin (IL)-6 secretion, although cisplatin did result in high induction. Moreover, BBI608 and SSZ, as other CSC-targeting drugs, could not suppress the expression of stemness markers. Together, Nanog expression appears related to poor prognosis in esophageal cancer patients, and inhibition of stemness and compensatory IL-6 secretion by iron chelators may offer a novel therapeutic strategy for esophageal cancer.
Keywords: esophageal cancer, cancer stem cells, stemness, iron, chelator
Abbreviations: 5-FU: 5-fluorouracil; CDDP: cisplatin; CIs: confidence intervals; CSCs: cancer stem cells; DFS: disease-free survival; DFX: deferasirox; ELISA: enzyme-linked immunosorbent assay; EMR: endoscopic mucosal resection; ESD: endoscopic submucosal dissection; HR: hazard ratio; HRs: Hazard ratios; IHC: immunohistochemistry; IL-6: interleukin-6; IL-6R: interleukin-6 receptor; miPS-LLCcm: mouse induced pluripotent stem cells epigenetically induced byLewis lung carcinoma conditioned medium; OS: overall survival; PBS: phosphate-buffered saline; PCR:
polymerase chain reaction; sIL-6R: soluble interleukin-6 receptor; SSZ: sulfasalazine; xCT:
cystine/glutamic acid transporter
Article category: Cancer therapy and prevention
Novelty and impact: High Nanog expression correlated with low OS only in the neoadjuvant therapy group of esophageal cancer patients. Nanog expression is a marker resistant to conventional 5-FU and/or CDDP chemotherapy. Iron chelators could suppress the expression of stemness markers including Nanog without compensatory IL-6 secretion, although 5-FU, CDDP and other CSC-targeting drugs such as SSZ and BBI608 could not suppress them. Iron chelators may offer a novel therapeutic strategy for esophageal cancer.
Introduction
Esophageal cancer is an important fatal malignancy all around the world. The 5-year survival rate is 10%, and the 5-year post-esophagectomy survival rate is 15–40% 1, 2. Although combination chemotherapy with 5-fluorouracil (5-FU) and cisplatin (CDDP) is standard for unresectable esophageal cancer, the response rate is only 35% 3. According to the cancer stem cell (CSC) hypothesis, cancer tissues have CSCs that are responsible for poor prognosis through chemoresistance and recurrence 4, 5. Some biomarkers of prognosis in esophageal cancer have been explored. Interleukin (IL)-6, a major inflammatory cytokine, reportedly correlates with poor
prognosis in esophageal cancer 6-8. However, secure biomarkers specifically related to CSCs have yet to be established 9-11.
Our previous study revealed that CSCs are highly dependent on iron for both proliferation and stemness using an induced pluripotent stem cell-derived CSC model (miPS-LLCcm) 12. Our previous study revealed that expression of Nanog was strongly regulated by iron chelators, suggesting that Nanog expression could be a useful biomarker for CSC-targeted treatment using iron chelators. However, whether CSC-targeted treatment and Nanog expression are clinically useful against esophageal cancer remains unclear. This study evaluated whether Nanog expression is a valuable biomarker in esophageal cancer patients, and examined the utility of CSC-targeted therapy by iron chelators compared with other CSC-targeted drugs for esophageal cancer.
Materials and methods Cell lines and cell culture
The TE8 (RRID:CVCL_1766) human esophageal squamous cancer cell line was purchased from RIKEN BRC Cell Bank (Tsukuba, Japan). The OE33 (RRID:CVCL_0471) human esophageal adenocarcinoma cell line was purchased from The European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK). All cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. TE8 and OE33 cells were maintained in RPMI containing 10% fetal calf serum (FCS), 50 U/ml of penicillin and 50 U/ml of streptomycin. All cell lines were recently authenticated using short tandem repeat profiling (October 2020) and all experiments were performed with mycoplasma-free cells.
Reagents
Deferasirox (DFX; EXJADE) and deferoxamine (DFO; Desferal) were obtained from Novartis Pharma (Tokyo, Japan). For in vitro studies, DFX was dissolved in dimethyl sulfoxide (Sigma-Aldrich, St Louis, MO, USA) at a stock concentration of 50 mM, and DFO was dissolved in distilled water at a stock concentration of 50 mM. Cisplatin (CDDP; Randa) was purchased from Nippon Kayaku (Tokyo, Japan) and dissolved in phosphate-buffered saline (PBS). The 5-fluorouracil (5-FU) was purchased from Kyowa Kirin (Tokyo, Japan), and dissolved in distilled water. SP was kindly provided by Disease Adsorption System Technologies Co., Ltd. (Kanazawa, Japan), and was chemically synthesized from catechol and benzoic acids. SP10 was made by a condensation reaction with histidine13, then dissolved in distilled water at the indicated concentrations for in vitro and in vivo experiments. Napabucasin (BBI608; 2-acetyl-4H,9H-naphtho[2,3-b] furan-4,9-dione) was purchased from Tokyo Kasei (Tokyo, Japan) and dissolved in dimethyl sulfoxide at a stock concentration of 50 mM. Sulfasalazine (SSZ) was purchased from FUJIFILM Wako Pure Chemical
Corporation (Osaka, Japan) and dissolved in dimethyl sulfoxide at the indicated concentrations.
IHC of clinical specimens
Tissue blocks of formalin-fixed,paraffin-embedded surgical specimens of esophageal cancer were sectioned into 2-mm slices for IHC. First, the presence of tumor was confirmed using hematoxylin and eosin (HE) staining. Sectioned tissues were then deparaffinized and soaked in 0.3% H2O2 in methanol at room temperature for 10 min to extinguish endogenous peroxidase activity. Antigen retrieval was performed by heating specimens in a sodium citrate buffer solution using a microwave.
After cooling, sectioned tissues were incubated with primary antibody against Nanog (catalog no.
4903S; Cell Signaling Technology, Danvers, MA, USA) overnight. After washing, the enzyme substrate 3,3'-diaminobenzidine (Agilent, Santa Clara, CA, USA) was used for visualization, and sections were counterstained with Meyer's hematoxylin. Staining intensity and the percentage of positive cells were scored according to previous studies (Supplementary Figure S1) 14, 15. The intensity of staining was determined as: 0=no staining; 1=weak staining; 2=moderate staining; and 3=strong staining (Supplementary Figure S1). The percentage of positive cells was scored as:
0=negative; 1=positive staining in ≤ 25% of tumor cells; 2=positive staining in 26–50% of tumor cells; 3=positive staining in 51–75% of tumor cells; and 4=positive staining in 76–100% of tumor cells. The two scores were multiplied and defined as: 0–4, low expression; and >4, high expression.
Cell viability assay
The XTT assay (Cell Proliferation kit II; Roche, Mannheim, Germany) was used to assess cell proliferation according to the protocol from the manufacturer. Cells were seeded on 96-well plates and treated with DFX, DFO, SP10, CDDP and 5-FU for 48 h at 37°C. We simulated iron-depleted conditions using iron-free RPMI plus 3% FCS.
Western blotting
Protein was extracted from whole cells after 48 h of incubation with medium and reagents.
Concentrations of extracted protein were measured using standard protocols. Cells were lysed using cell lysis buffer (50 mmol/L Tris-HCl (pH 7.4), 30 mmol/L NaCl, and 1% Triton X-100) containing protease inhibitors (cOmplete Mini; Roche Diagnostics GmbH, Basel, Switzerland). Equal amounts of total cellular proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred electrophoretically to polyvinylidene difluoride filter membranes (GE Healthcare UK, Buckinghamshire, UK). Protein samples were separated on tris-acrylamide gels and transferred to PVDF membranes (GE Healthcare Life Sciences) according to the protocols of the manufacturer. The following primary antibodies were used: anti-Nanog antibody (catalog no. 4903S;
Cell Signaling Technology), anti-SOX2 antibody (catalog no. ab97959; Abcam, Cambridge, MA,
USA), anti-Oct4 antibody (catalog no. ab18976; Abcam), anti-KLF4 antibody (catalog no. 4038S;
Cell Signaling Technology), anti-c-Myc antibody (catalog no. 5605S; Cell Signaling Technology), anti-STAT3 antibody (catalog no. 12640S; Cell Signaling Technology), anti-pSTAT3 antibody (catalog no. 9145S; Cell Signaling Technology), anti-β-actin antibody (catalog no. A5441, Sigma-Aldrich). All primary antibodies were used at a 1:1000 dilution. The following secondary antibodies were used: anti-mouse immunoglobulin (Ig)G, horseradish peroxidase (HRP)-linked whole sheep antibody (catalog no. NA931; GE Healthcare UK), anti-rabbit IgG, HRP-linked whole donkey antibody (catalog no. NA934; GE Healthcare UK). All secondary antibodies (GE Life Sciences) were used at a 1:2500 dilution. Membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies. ECL prime Western Blotting Detection Reagent (GE Healthcare UK) was used to detect the peroxidase activity of secondary antibodies. Membranes were probed for β-actin as a loading control, and all sample data values were normalized to the corresponding control data values.
RNA microarray analysis
TE8 cells were seeded at 1 ×105 cells/mL in a 10-cm Petri dish 24 h before treatment of CDDP at 10 µM for 48 h. Total RNA was isolated from TE8 cells using a High Pure RNA Isolation kit (Roche Applied Science, Basel, Switzerland), respectively. RNA microarray analysis was performed using the SurePrint G3 Human Gene Expression 8 × 60k version 3.0 as contract analysis (Macrogen Japan, Kyoto, Japan). The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus (Ohara et al., 2021) and are accessible through GEO Series accession number GSE164201 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE164201).
IL-6 ELISA assay
To evaluate supernatant IL-6 secreted by TE8 and OE33 cells, we used an IL-6 ELISA kit (Biolegend, San Diego, CA, USA). Cancer cells were plated in T25 flasks and treated with different concentrations of DFX, SP10, CDDP, and 5-FU. After treatment for 48 h, supernatant was harvested and the IL-6 content was assayed by ELISA according to the protocol provided by the manufacturer.
Real-time quantitative PCR
TE8 and OE33 cells were seeded at 5 ×104 cells/mL in 6-well plates 24 h before treatment with different concentrations of DFX, SP10, CDDP, and 5-FU for 48 h. Total RNA was isolated from TE8 and OE33 cells using a High Pure RNA Isolation kit (Roche Applied Science). First-strand cDNA was constructed from total RNA using the oligo-(dT) primer. Real-time quantitative PCR analysis was performed using StepOne with Taqman PCR master mix (Applied Biosystems, Foster City, CA, USA). The primers used in this study were: GAPDH (Applied Biosystems) and IL-6 (Integrated
DNA Technologies, Coralville, IA, USA). Quantification of the gene of interest was normalized to GAPDH and expressed as fold-increases relative to the negative control for each treatment at each time point, as previously described.
Tumor xenograft model and experiment
Eight-week-old female BALB/c (nu/nu) mice were purchased from CLEA Japan (Tokyo, Japan).
TE8 cells in culture were harvested and resuspended in a 1:1 ratio of PBS and Matrigel (BD Biosciences, San Jose, CA, USA). TE8 (3.0 ×106 cells) were injected subcutaneously into the backs of mice. After 7 days, tumor-bearing animals were randomized into two groups of 4 mice each: i) SP10 (200 mg⁄kg administered intraperitoneally 5 times/week for 2 weeks); and ii) saline alone as a vehicle (given intraperitoneally 5 times/week for 2 weeks). Tumor size and body weight were measured. Tumor volume (mm3) was calculated as length × width × height × π/6. At the end of the experiment, mice were sacrificed and tumors were excised, weighed, and processed for histological analysis.
Immunohistochemistry of in vivo-derived tumor tissues
Harvested tumors were fixed in 10% paraformaldehyde and embedded in paraffin prior to immunostaining analysis. Anti-Nanog antibody was used as in Western blot analysis.
Statistical analysis
All analyses were performed using EZR ver.1.40 (Saitama Medical Center, Jichi Medical University, Saitama, Japan)16. Overall survival (OS) and disease-free survival (DFS) were calculated using Kaplan-Meier methods, and the log-rank test was used for comparisons between subgroups. Hazard ratios (HRs) and 95% confidence intervals (CIs) for clinical variables were calculated using Cox proportional hazards regression in uni- and multivariate analyses. The χ2 test of independence was used to analyze patient characteristics. For two-group comparisons, Student’s t-test was used. In multi-group analyses, one-way analysis of variance followed by the Dunnett test was used. Values of p < 0.05 were considered statistically significant.
Results
High Nanog expression correlates with poor prognosis in esophageal cancer patients
First, to verify the association of Nanog expression with poor prognosis in esophageal cancer patients, immunohistochemistry (IHC) for Nanog was performed. A total of 149 esophageal cancer patients who underwent radical esophagectomy in the Department of Gastroenterological Surgery at Okayama University Hospital between 2008 and 2010 were registered for this study. Fifteen cases were excluded after undergoing endoscopic mucosal resection (EMR) or endoscopic submucosal
dissection (ESD) followed by surgery, diagnosis with melanoma or distant metastasis, no tumor presence due to complete response after neo-adjuvant therapy, or irrelevant formalin fixation of specimens (Figure 1A). Nanog expression was scored as described in previous studies (Supplementary Figure S1) 14, 15. In 134 cases, Nanog expression was high in 98 cases and low in 36 cases. Clinicopathological features are shown in Table 1. Interestingly, no correlation was apparent between Nanog expression and tumor progression (T, N, and clinical stage). Analysis of the Kaplan-Meier curve revealed that high Nanog expression correlated with low OS (HR, 2.53; 95%CI, 1.24–5.14; P = 0.01) and low DFS (HR, 2.33; 95%CI, 1.19–4.58; P = 0.01) (Figure 1B, 1C). Both uni- and multivariate Cox regression analyses identified high Nanog expression as associated with poor prognosis (Supplementary Tables 1, 2). These results indicate that Nanog expression may offer a valuable biomarker for prognosis in esophageal cancer patients.
High Nanog expression correlates with chemoresistance to neoadjuvant therapy
Next, to explore the reasons for poor prognosis related to high expression of Nanog, we performed subgroup analyses of 134 cases, divided into groups with and without neoadjuvant therapy (Figure 2A). The results interestingly showed that high Nanog expression correlated with low OS in the neoadjuvant therapy group, but not in the non-neoadjuvant therapy group (Figure 2B, 2C). This finding suggests that Nanog expression correlates with resistance to conventional 5-FU and/or CDDP chemotherapy and may induce the overall poor prognosis in esophageal cancer patients.
Iron chelators suppress both cancer cell proliferation and stemness markers expression in esophageal cancer cell lines
To evaluate the effect of standard chemotherapy (CDDP + 5-FU) and iron chelators (DFX, SP10) on esophageal cancer cell lines, we examined cell viability and the expression of stemness markers employing esophageal squamous cell carcinoma (TE8) and adenocarcinoma (OE33) cell lines, which show high expression of stemness markers. XTT assay showed that standard chemotherapy and iron chelators suppressed proliferation in TE8 and OE33 cells (Figure 3A, 3C). Although expression of stemness markers was maintained or partially upregulated by standard chemotherapy, most stemness markers were downregulated only by iron chelators (Figure 3B, 3D). These results revealed that standard chemotherapy maintained or partially upregulated stemness markers, especially Nanog, which may induce poor prognosis. Conversely, iron chelators suppressed stemness marker expression, indicating the potential to improve the poor prognosis in esophageal cancer patients.
Iron chelator does not induce compensatory IL-6 secretion compared with CDDP and 5-FU The poor prognosis of esophageal cancer patients has been reported to be related to inflammation, particularly inflammatory cytokines. IL-6, a major inflammatory cytokine, reportedly correlates with
poor prognosis 6-8. We performed exploratory microRNA array analysis in TE8 cells to explore differences in the expression of inflammatory cytokines by CDDP. The microRNA array analysis revealed that expression of IL-6 and the IL-6 receptor (IL-6R) are elevated by CDDP, indicating that IL-6 expression was possibly elevated by standard chemotherapy in esophageal cancer cells (Figure 4A). We thus performed real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) of IL-6 to confirm compensatory induction and to explore the therapeutic potential of iron chelators. The real-time PCR revealed that both CDDP and 5-FU induced mRNA of IL-6, whereas iron chelators did not (Figure 4B). ELISA showed that CDDP dramatically induced IL-6 secretion. In contrast, iron chelators did not induce IL-6 secretion compared with CDDP (Figure 4C). Similarly, 5-FU did not induce significant IL-6 secretion. These results suggested that CDDP, as a standard chemotherapy agent, induced compensatory IL-6 secretion and thus possibly correlated with poor prognosis in esophageal cancer through inflammation. These results also suggested that iron chelators have the potential to improve the prognosis by avoiding compensatory IL-6 secretion.
Stemness marker suppression is a specific and superior effect of iron chelators as a CSC-targeting drug
To evaluate performance as a CSC-targeting drug, we compared DFX and SP10 with napabucasin and sulfasalazine as other CSC-targeting drugs. Napabucasin (BBI608) targets CSCs by inhibiting STAT3 signaling. Sulfasalazine (SSZ) also targets CD44-positive CSCs by inducing ferroptosis via suppression of cystine/glutamic acid transporter (xCT). These drugs have already been applied for clinical studies. XTT assay showed that both BBI608 and SSZ suppressed proliferation of TE8 and OE33 cells (Figure 5A). However, neither BBI608 nor SSZ could suppress the expression of stemness markers (Nanog, c-myc, and Klf4) (Figure 5B). These results indicated that suppression of stemness markers is a specific and superior effect of iron chelators as CSC-targeted drugs. In addition, we examined the anti-tumor effects of SP10 using a subcutaneous xenograft model of TE8.
SP10 is a newly developed iron chelator and is expected to show few side effects compared with other iron chelators. SP10 significantly inhibited tumor growth and the suppression of Nanog in tumor tissue was confirmed by IHC (Figure 5C, 5D).
Discussion
In the 134 immunohistochemical analyzed cases, high Nanog expression correlated with both low OS and low DFS. No significant deviations in tumor depth, lymph node metastasis, clinical stage, or neoadjuvant therapy were identified according to Nanog expression. However, Nanog expression correlated significantly with low OS and low DFS only in the neoadjuvant therapy group, suggesting that Nanog works as a chemo-resistant marker. The regimen of chemotherapy used in this study was
almost CDDP and 5-FU, indicating that some mechanisms involving Nanog are responsible for the resistance conferred by CDDP or 5-FU. This finding was helpful to identify the compensatory induction of IL-6 by CDDP. Poor prognosis among esophageal cancer patients reportedly correlates with Nanog expression 14. However, this is the first report to identify that poor prognosis correlated strongly with neoadjuvant therapy. Moreover, this finding is worthy because the present standard protocol for resectable esophageal cancer is neoadjuvant chemotherapy, and comparison with a no-neoadjuvant therapy group is thus extremely difficult.
We examined the mechanisms of stemness maintenance through IL-6 regulation. As one of the important inflammatory cytokines, IL-6 signals through IL-6R and the signal-transducing component gp130 to activate the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway. The gp130 protein is a common signal transducer for several cytokines, including leukemia inhibitory factor (LIF). We thus evaluated stemness regulation by IL-6 and LIF recombinant protein. IL-6 enhanced the proliferation of TE8 and OE33 (Supplementary Figure S3A). Neither IL-6 nor IL-6 combined with soluble IL-6 receptor (sIL-6R) enhanced expression of Nanog (Supplementary Figure S3B). LIF also did not enhance the proliferation and expression of Nanog (Supplementary Figure S3C, D). Moreover, anti-IL-6 antibody and anti-LIF antibody were confirmed to not suppress the expression of Nanog (Supplementary Figure 3E). These results suggest that IL-6 does not regulate stemness directly via autocrine mechanisms. However, we cannot completely rule out the regulation of stemness by IL-6 signaling, as IL-6 may indirectly regulate stemness through some kinds of cells, such as cancer-associated fibroblasts and tumor-associated macrophages in the tumor microenvironment 17-23. Moreover, some enzymes and non-coding RNAs have recently been reported to be related with stemness regulation in esophageal cancer 24, 25. Further examinations of stemness regulation focusing on iron in the tumor microenvironment are thus needed in the near future.
The characteristics of CSCs are often expressed by expression of stemness markers and key signal pathways. Some stemness markers, including Nanog, and key signal pathways such as Wnt/β-catenin, Notch and JAK/STAT3 are known as characteristics of CSCs in esophageal cancer 5,
26. These show partial overlap with other cancers 27-29. Nanog is reportedly expressed in other gastrointestinal cancers and is known as an important stemness marker to acquire stemness potential
29-31. No CSC-targeting drugs have been established, although many candidate drugs have been developed. BBI608 (napabucasin) was developed to target CSCs via inhibition of STAT3 signaling 32,
33. However, this agent failed to suppress the expressions of Nanog, c-Myc, or Klf4, even though STAT3 signaling correlated with expressions of these stemness markers 34. SSZ was developed to target CD44-positive CSCs via suppression of xCT 35, 36. CD44 is known as a common CSC marker in several cancers, including esophageal cancer 29, 37, 38. CD44 has been reported to interact with Nanog through ERK1/2 and β-catenin pathways 39, 40. These candidate drugs for CSCs could not
suppress stemness potential, at least in esophageal cancer. Here it is appropriate to discuss stemness suppression by iron chelators. Many iron chelators have the ability to suppress the expression of stemness markers, including in cholangiocarcinoma, although we revealed the result of DFX, SP10, and DFO in some esophageal cancer cell lines 41 (Figure 3, Supplementary Figure S2A, S2B, S2C).
SSZ is also a kind of iron chelator and is able to bind xCT. All iron chelators in this study were confirmed to chelate iron ions (Supplementary Figure S4). This result suggested that not all iron chelators have the ability to suppress the expression of stemness markers. However, iron chelators are promising drugs to regulate stemness as cancer therapy. A more detailed understanding of the mechanisms by which iron chelators suppress stemness in cells should thus be explored, and may yield significant progress in CSC-targeting research.
The present study has the following limitations. The relationship between Nanog expression and IL-6 remains unclear for esophageal cancer, although we ruled out direct regulation via autocrine mechanisms. The safety of SP10 is still unclear, although SP10 was developed to be safer than other chelators 42. Further studies are needed to evaluate the effects of iron chelators on esophageal cancer.
In conclusion, CSCs expressing Nanog may contribute to the poor prognosis of esophageal cancer patients. Inhibition of stemness and compensatory IL-6 secretion by iron chelators can be a novel therapeutic strategy for esophageal cancer.
Conflicts of interest: The authors have no conflicts of interest to declare.
Author contributions: Conceptualization: T. Ohara, K. Noma, and T. Fujiwara; investigation: T.
Narusaka, T. Ohara, N. Nishiwaki, Y. Katsura, and T. Kato; animal investigation: Y. Tomono;
resources: A. Matsukawa and T. Fujiwara; writing-original graft preparation: T. Narusaka; review and editing: T. Ohara; supervision: K. Noma, S. Kikuchi, H. Tazawa, Y. Shirakawa, A. Matsukawa, and T. Fujiwara; funding acquisition: T. Ohara
Acknowledgements
We are grateful to Tomoko Sueishi, Tae Yamanishi, and Shiho Komaki for their kind assistance with the in vitro and in vivo experiments. This work was supported by grants-in-aid from the Ministry of Education, Science, and Culture, Japan; and grants from the Ministry of Health and Welfare, Japan (18K08539).
Data availability statement
The RNA microarray data generated in this study is available in GEO under accession GSE164201 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE164201). Other data are available from
the corresponding author upon request.
Ethics statement
The use of pathologic samples was approved and reviewed by the Ethics Review Board of Okayama University, Okayama, Japan (No. 1801–023). Details have been removed from these case descriptions to ensure anonymity. All animal experiments were performed according to the Japanese Welfare and Management of Animals Act and conducted in accordance with institutional guidelines at Shigei Medical Research Institute, Okayama, Japan. All animal experiments were approved by the Ethics Review Committee for Animal Experimentation of Shigei Medical Research Institute (#190401-1), Okayama, Japan.
REFERENCES
1. Huang FL, Yu SJ. Esophageal cancer: Risk factors, genetic association, and treatment.
Asian J Surg 2018;41:210-15.
2. Kato H, Nakajima M. Treatments for esophageal cancer: a review. Gen Thorac Cardiovasc Surg 2013;61:330-5.
3. Kitagawa Y, Uno T, Oyama T, Kato K, Kato H, Kawakubo H, Kawamura O, Kusano M, Kuwano H, Takeuchi H, Toh Y, Doki Y, et al. Esophageal cancer practice guidelines 2017 edited by the Japan esophageal society: part 2. Esophagus 2019;16:25-43.
4. Valent P, Bonnet D, De Maria R, Lapidot T, Copland M, Melo JV, Chomienne C, Ishikawa F, Schuringa JJ, Stassi G, Huntly B, Herrmann H, et al. Cancer stem cell definitions and terminology: the devil is in the details. Nat Rev Cancer 2012;12:767-75.
5. Wu Q, Wu Z, Bao C, Li W, He H, Sun Y, Chen Z, Zhang H, Ning Z. Cancer stem cells in esophageal squamous cell cancer. Oncol Lett 2019;18:5022-32.
6. Groblewska M, Mroczko B, Sosnowska D, Szmitkowski M. Interleukin 6 and C-reactive protein in esophageal cancer. Clin Chim Acta 2012;413:1583-90.
7. Dvorak K, Dvorak B. Role of interleukin-6 in Barrett's esophagus pathogenesis.
World J Gastroenterol 2013;19:2307-12.
8. Maeda Y, Takeuchi H, Matsuda S, Okamura A, Fukuda K, Miyasho T, Nakamura R, Suda K, Wada N, Kawakubo H, Kitagawa Y. Clinical significance of preoperative serum concentrations of interleukin-6 as a prognostic marker in patients with esophageal cancer.
Esophagus 2020;17:279-88.
9. Zhao L, Liu J, Chen S, Fang C, Zhang X, Luo Z. Prognostic significance of NANOG expression in solid tumors: a meta-analysis. Onco Targets Ther 2018;11:5515-26.
10. Liang C, Zhao T, Ge H, Xu Y, Ren S, Yue C, Li G, Wu J. The clinicopathological and prognostic value of Nanog in human gastrointestinal luminal cancer: A meta-analysis. Int J
Surg 2018;53:193-200.
11. Yu SS, Cirillo N. The molecular markers of cancer stem cells in head and neck tumors. J Cell Physiol 2020;235:65-73.
12. Ninomiya T, Ohara T, Noma K, Katsura Y, Katsube R, Kashima H, Kato T, Tomono Y, Tazawa H, Kagawa S, Shirakawa Y, Kimura F, et al. Iron depletion is a novel therapeutic strategy to target cancer stem cells. Oncotarget 2017;8:98405-16.
13. Nishida Y. The chemical mechanism of oxidative stress due to the non-transferrin-bound iron (NTBI). Advances in Bioscience and Biotechnology 2012;3:1076-86.
14. Sun LL, Wu JY, Wu ZY, Shen JH, Xu XE, Chen B, Wang SH, Li EM, Xu LY. A three-gene signature and clinical outcome in esophageal squamous cell carcinoma. Int J Cancer 2015;136:E569-77.
15. Tamaki T, Shimizu T, Niki M, Shimizu M, Nishizawa T, Nomura S.
Immunohistochemical analysis of NANOG expression and epithelial-mesenchymal transition in pulmonary sarcomatoid carcinoma. Oncol Lett 2017;13:3695-702.
16. Kanda Y. Investigation of the freely available easy-to-use software 'EZR' for medical statistics. Bone Marrow Transplant 2013;48:452-8.
17. Batlle E, Clevers H. Cancer stem cells revisited. Nature medicine 2017;23:1124-34.
18. Kato T, Noma K, Ohara T, Kashima H, Katsura Y, Sato H, Komoto S, Katsube R, Ninomiya T, Tazawa H, Shirakawa Y, Fujiwara T. Cancer-Associated Fibroblasts Affect Intratumoral CD8(+) and FoxP3(+) T Cells Via IL6 in the Tumor Microenvironment. Clinical cancer research : an official journal of the American Association for Cancer Research 2018;24:4820-33.
19. Liu ML, Zang F, Zhang SJ. RBCK1 contributes to chemoresistance and stemness in colorectal cancer (CRC). Biomed Pharmacother 2019;118:109250.
20. Huang TX, Guan XY, Fu L. Therapeutic targeting of the crosstalk between cancer-associated fibroblasts and cancer stem cells. Am J Cancer Res 2019;9:1889-904.
21. Chen JH, Wu ATH, Bamodu OA, Yadav VK, Chao TY, Tzeng YM, Mukhopadhyay D, Hsiao M, Lee JC. Ovatodiolide Suppresses Oral Cancer Malignancy by Down-Regulating Exosomal Mir-21/STAT3/beta-Catenin Cargo and Preventing Oncogenic Transformation of Normal Gingival Fibroblasts. Cancers (Basel) 2019;12.
22. Chan TS, Shaked Y, Tsai KK. Targeting the Interplay Between Cancer Fibroblasts, Mesenchymal Stem Cells, and Cancer Stem Cells in Desmoplastic Cancers. Front Oncol 2019;9:688.
23. Penn CA, Yang K, Zong H, Lim JY, Cole A, Yang D, Baker J, Goonewardena SN, Buckanovich RJ. Therapeutic Impact of Nanoparticle Therapy Targeting Tumor-Associated Macrophages. Mol Cancer Ther 2018;17:96-106.
24. Tan Y, Lu X, Cheng Z, Pan G, Liu S, Apiziaji P, Wang H, Zhang J, Abulimiti Y.
miR-148a Regulates the Stem Cell-Like Side Populations Distribution by Affecting the Expression of ACVR1 in Esophageal Squamous Cell Carcinoma. Onco Targets Ther 2020;13:8079-94.
25. Qian Y, Liang X, Kong P, Cheng Y, Cui H, Yan T, Wang J, Zhang L, Liu Y, Guo S, Cheng X, Cui Y. Elevated DHODH expression promotes cell proliferation via stabilizing beta-catenin in esophageal squamous cell carcinoma. Cell Death Dis 2020;11:862.
26. Zhou C, Fan N, Liu F, Fang N, Plum PS, Thieme R, Gockel I, Gromnitza S, Hillmer AM, Chon SH, Schlosser HA, Bruns CJ, et al. Linking Cancer Stem Cell Plasticity to Therapeutic Resistance-Mechanism and Novel Therapeutic Strategies in Esophageal Cancer. Cells 2020;9.
27. Korourian A, Roudi R, Shariftabrizi A, Kalantari E, Sotoodeh K, Madjd Z.
Differential role of Wnt signaling and base excision repair pathways in gastric adenocarcinoma aggressiveness. Clin Exp Med 2017;17:505-17.
28. Saeednejad Zanjani L, Madjd Z, Rasti A, Asgari M, Abolhasani M, Tam KJ, Roudi R, Maelandsmo GM, Fodstad O, Andersson Y. Spheroid-Derived Cells From Renal Adenocarcinoma Have Low Telomerase Activity and High Stem-Like and Invasive Characteristics. Front Oncol 2019;9:1302.
29. Walcher L, Kistenmacher AK, Suo H, Kitte R, Dluczek S, Strauss A, Blaudszun AR, Yevsa T, Fricke S, Kossatz-Boehlert U. Cancer Stem Cells-Origins and Biomarkers: Perspectives for Targeted Personalized Therapies. Front Immunol 2020;11:1280.
30. Najafzadeh B, Asadzadeh Z, Motafakker Azad R, Mokhtarzadeh A, Baghbanzadeh A, Alemohammad H, Abdoli Shadbad M, Vasefifar P, Najafi S, Baradaran B. The oncogenic potential of NANOG: An important cancer induction mediator. J Cell Physiol 2020.
31. Katsura Y, Ohara T, Noma K, Ninomiya T, Kashima H, Kato T, Sato H, Komoto S, Narusaka T, Tomono Y, Xing B, Chen Y, et al. A Novel Combination Cancer Therapy with Iron Chelator Targeting Cancer Stem Cells via Suppressing Stemness. Cancers (Basel) 2019;11.
32. Tan BT, Park CY, Ailles LE, Weissman IL. The cancer stem cell hypothesis: a work in progress. Lab Invest 2006;86:1203-7.
33. Kawazoe A, Kuboki Y, Bando H, Fukuoka S, Kojima T, Naito Y, Iino S, Yodo Y, Doi T, Shitara K, Yoshino T. Phase 1 study of napabucasin, a cancer stemness inhibitor, in patients with advanced solid tumors. Cancer Chemother Pharmacol 2020;85:855-62.
34. Wang ML, Chiou SH, Wu CW. Targeting cancer stem cells: emerging role of Nanog transcription factor. Onco Targets Ther 2013;6:1207-20.
35. Nagano O, Okazaki S, Saya H. Redox regulation in stem-like cancer cells by CD44 variant isoforms. Oncogene 2013;32:5191-8.
36. Shitara K, Doi T, Nagano O, Imamura CK, Ozeki T, Ishii Y, Tsuchihashi K,
Takahashi S, Nakajima TE, Hironaka S, Fukutani M, Hasegawa H, et al. Dose-escalation study for the targeting of CD44v(+) cancer stem cells by sulfasalazine in patients with advanced gastric cancer (EPOC1205). Gastric Cancer 2017;20:341-49.
37. Okamoto K, Ninomiya I, Ohbatake Y, Hirose A, Tsukada T, Nakanuma S, Sakai S, Kinoshita J, Makino I, Nakamura K, Hayashi H, Oyama K, et al. Expression status of CD44 and CD133 as a prognostic marker in esophageal squamous cell carcinoma treated with neoadjuvant chemotherapy followed by radical esophagectomy. Oncol Rep 2016;36:3333-42.
38. Kamil Mohammed Al-Mosawi A, Cheshomi H, Hosseinzadeh A, M MM. Prognostic and Clinical Value of CD44 and CD133 in Esophageal Cancer: A Systematic Review and Meta-analysis. Iran J Allergy Asthma Immunol 2020;19:105-16.
39. Radwan AA, Al-Mohanna F, Alanazi FK, Manogaran PS, Al-Dhfyan A. Target beta-catenin/CD44/Nanog axis in colon cancer cells by certain N'-(2-oxoindolin-3-ylidene)-2-(benzyloxy)benzohydrazides. Bioorg Med Chem Lett 2016;26:1664-70.
40. Huang C, Yoon C, Zhou XH, Zhou YC, Zhou WW, Liu H, Yang X, Lu J, Lee SY, Huang K. ERK1/2-Nanog signaling pathway enhances CD44(+) cancer stem-like cell phenotypes and epithelial-to-mesenchymal transition in head and neck squamous cell carcinomas.
Cell Death Dis 2020;11:266.
41. Raggi C, Gammella E, Correnti M, Buratti P, Forti E, Andersen JB, Alpini G, Glaser S, Alvaro D, Invernizzi P, Cairo G, Recalcati S. Dysregulation of Iron Metabolism in Cholangiocarcinoma Stem-like Cells. Scientific reports 2017;7:17667.
42. Ohara T, Tomono Y, Boyi X, Yingfu S, Omori K, Matsukawa A. A novel, nontoxic iron chelator, super-polyphenol, effectively induces apoptosis in human cancer cell lines.
Oncotarget 2018;9:32751-60.
Figure Legends
Figure 1. Correlation between Nanog expression and clinical outcome in 134 cases of esophageal cancer.
(A) Diagram of esophageal cancer patients who underwent Nanog IHC.
(B) OS curves according to Nanog expression (high or low group) in the intratumoral tissues. The high Nanog expression group showed significantly poor prognosis (Cox regression hazard model, 95% confidence intervals (CIs), and log-rank test).
(C) DFS curves according to Nanog expression (high or low groups) in the intratumoral tissues. The high Nanog expression group showed significantly poor prognosis (Cox regression hazard model, 95%CI, and log-rank test).
Figure 2. Subgroup analysis of 134 cases in esophageal cancer.
(A) Diagram of subgroup analysis for esophageal cancer patients who underwent Nanog IHC. (B) OS curves according to Nanog expression (high or low group) in intratumoral tissues. High Nanog expression correlated with significantly poorer prognosis in the neoadjuvant therapy group (log-rank test). (C) OS curves according to Nanog expression (high or low group) in intratumoral tissues. High Nanog expression did not correlate with significantly poorer prognosis in the non-neoadjuvant therapy group (log-rank test).
Figure 3. Effect of iron chelators on proliferation and expression of stemness markers in human esophageal cancer cell lines in vitro.
(A) Cultured TE8 cells and OE33 cells were treated with different concentrations of CDDP or 5-FU for 48 h, and cell viability was evaluated with the XTT assay. Both drugs suppressed proliferation of TE8 cells and OE33 cells in a dose-dependent manner. Cell viability in the absence of treatment was set at 100%. *p < 0.05, **p < 0.01. (B) After culturing TE8 cells and OE33 cells with different concentrations of CDDP or 5-FU for 48 h, cell lysates were collected, and total protein was analyzed for expression of the indicated stemness markers using western blot analysis. Expression of stemness markers was not suppressed by CDDP or 5-FU. (C) Cultured TE8 cells and OE33 cells were treated with different concentrations of DFX or SP10 for 48 h, and cell viability was evaluated with the XTT assay. DFX and SP10 suppressed the proliferation of TE8 cells and OE33 cells in a dose-dependent manner. Cell viability in the absence of treatment was set at 100%. *p < 0.05, **p <
0.01. (D) After culturing TE8 cells and OE33 cells with different concentrations of DFX or SP10 for 48 h, cell lysates were collected, and total protein was analyzed for expression of the indicated stemness markers with Western blot analysis. Most stemness markers were suppressed after treatment with DFX or SP10.
Figure 4. Iron chelators did not induce autocrine IL-6 compared with conventional chemotherapy.
(A) Analysis by RNA microarray revealed expression levels of each cytokine in TE8 cells treated with CDDP. IL-6 expression level was increased compared with other cytokines. (B) Cultured TE8 cells and OE33 cells were treated with different concentrations of CDDP, 5-FU, DFX, or SP10 for 48 h, and IL-6 mRNA expression levels were evaluated with real-time PCR. Conventional chemotherapeutic drugs induced IL-6 mRNA. (C) Cultured TE8 cells and OE33 cells were treated with different concentrations of CDDP, 5-FU, DFX, or SP10 for 48 h, and IL-6 autocrine levels were evaluated by ELISA. CDDP induced IL-6 autocrine in a dose-dependent manner.
Figure 5. Effects of other CSC-targeting drugs on proliferation and expression of stemness
markers in vitro, and anti-tumor effects of SP10 in vivo.
(A) Cultured TE8 cells and OE33 cells were treated with different concentrations of BBI608 or SSZ for 48 h, and cell viability was evaluated using the XTT assay. BBI608 and SSZ suppressed the proliferation of TE8 cells and OE33 cells in a dose-dependent manner. Cell viability in the absence of treatment was set at 100%. *p < 0.01. (B) After culturing TE8 cells and OE33 cells with different concentrations of BBI608 or SSZ for 48 h, cell lysates were collected, and total protein was analyzed for expression of the indicated stemness markers in Western blot analysis. Phospho-Stat3 was confirmed to be suppressed by BBI608. Expression of stemness markers (Nanog, c-Myc and Klf4) was not suppressed by BBI608 and SSZ. (C) TE8 cells were injected subcutaneously into the dorsal skin of mice. After 7 days, mice were randomly dived into two groups (n = 4 each group), and treatment was initiated as indicated. SP10 effectively inhibited the growth of TE8 xenograft in vivo.
*p < 0.05. (D) Resected tumors were analyzed for Nanog expression by IHC. Nanog was suppressed in the SP10 groups.
Table 1. Clinicopathological characteristics of esophageal cancer patients who underwent Nanog IHC.
Nanog expression
Total High Low p value*
Age, years
<67 64 50 14
0.293
≥67 70 48 22
Sex
Male 109 84 27
0.230
Female 25 14 9
Pathology
Squamous cell carcinoma 115 87 28
0.181
Adenocarcinoma or other 19 11 8
T
T1 61 44 17
0.965
T2 or greater 73 54 19
N
N0 63 43 21
0.197
N1 or greater 68 55 15
Clinical stage
0 to I 47 32 15
0.444
II or greater 87 66 21
Neoadjuvant therapy (CT or CRT)
Performed 32 27 5
0.157
Not performed 102 71 31
*The χ2 test of independence was used to analyze patient characteristics.
