Elsevier

Oral Oncology

Volume 49, Issue 1, January 2013, Pages 34-41
Oral Oncology

Epithelial–mesenchymal transition transcription factor ZEB1/ZEB2 co-expression predicts poor prognosis and maintains tumor-initiating properties in head and neck cancer

https://doi.org/10.1016/j.oraloncology.2012.07.012Get rights and content

Summary

Objectives

Both epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties may be involved in metastasis, which contributes to the high mortality rate of patients with head and neck cancers (HNCs). However, the mechanisms through which the EMT transcription factors ZEB1 and ZEB2 regulate HNC are still unclear.

Methods

Tumor initiating capability of HNC-CH133+ cells with ZEB1/2 knockdown or co-overexpression was presented in vitro and in vivo.

Results

In the present study, we demonstrated that ZEB1/ZEB2 expression was significantly increased in HNC-CD133+ CSC-like cells compared with HNC-CD133 cells. The small interfering RNA (siRNA)-mediated co-knockdown of ZEB1 and ZEB2 (siZEB1/2) in HNC-CH133+ cells suppressed their CSC-like properties, including self-renewal ability, the expression of stemness markers, and drug resistance. In contrast, the co-overexpression of ZEB1/ZEB2 in HNC-CD133 cells enhanced their sphere-forming ability and increased the percentage of CD44-positive cells and side population cells. In vivo studies showed that the delivery of siZEB1/2 to xenograft tumors in nude mice reduced tumor growth and the rate of distant metastasis. In clinical samples, the levels of ZEB1/ZEB2 expression were low in local lesions but high in metastatic lymph nodes in HNC tissues. Patients with tumors that co-expressed ZEB1high and ZEB2high had especially poor survival rates.

Conclusion

Therapies targeting ZEB1/ZEB2 in HNC-CD133+ cells may provide a new approach for HNC therapy in the future.

Introduction

Head and neck cancer (HNC) is the sixth most common malignancy worldwide and the third most common cancer in developing nations.1 The prevalence of HNC is also high in Taiwan due to the regional habit of areca nut chewing. HNC-related death is primarily caused by cervical lymph node metastasis and occasionally by distant organ metastasis.2 Despite improvements in the diagnosis and management of HNC, long-term survival rates have improved only marginally over the past decade.3 To improve the survival rate of HNC patients, investigations to elucidate the mechanism of the tumorigenesis of HNC are urgently needed.

Recent studies have suggested that the persistent survival of cancer stem cells (CSCs), also known as tumor-initiating cells (TICs), may contribute to the aggression of HNCs.4, 5, 6, 7 These CSCs are key contributors to radioresistance and are responsible for tumor progression and recurrence after conventional therapy.8 CD133 (prominin-1: PROM1), a 5-transmembrane glycoprotein, is a hematopoietic stem cell and endothelial progenitor marker that appears to be involved in angiogenesis.9 CD133 expression has been suggested to serve as a prognostic signature of tumor regrowth, malignancy progression, and tumor stages in leukemia, retinoblastoma, colon cancer, prostate cancer, brain tumor, and hepatoma.10 Some studies have reported that HNC-derived CD133+ cells display CSC-like properties, such as tumor initiation and chemoresistance.4, 11, 12, 13 Therefore, it is important to understand the biology of these cells and develop therapeutic strategies to target intractably malignant CD133+ HNC cells.

Epithelial–mesenchymal transition (EMT), a de-differentiation program that converts adherent epithelial cells to individual migratory cells, is critical for the enhancement of CSC traits.14 EMT is enhanced in lung cancers by the up-regulation of embryonic stem cell (ESC) genes, including Oct4 and Nanog, and results in increased tumor metastasis.15 However, EMT-related transcription factors promote stemness in normal breast tissue and breast cancer cells.16 ZEB1 and ZEB2, members of the zinc-finger E-box-binding homeobox factor (ZEB) family, are transcriptional repressors that contain two widely separated clusters of C2H2-type zinc fingers that mediate their binding to paired CAGGTA/GE-box-like promoter elements.17, 18 These repressors induce EMT by suppressing the expression of E-cadherin and contribute to the progression of malignant cancer.17, 18 ZEB1 and ZEB2 are good predictors of prognosis in bladder cancer, prostate cancer, brain cancer, breast cancer, pancreatic cancer, and lung cancer.19, 20, 21, 22, 23 These proteins have also been implicated in drug resistance and CSC properties in pancreatic cancer.17 However, the role of ZEB1- and ZEB2-mediated CSC properties in HNC remains unclear.

In the present study, we demonstrated that ZEB1 and ZEB2 expression was significantly elevated in HNC-CD133+ cells. We evaluated the effects of the co-knockdown or co-overexpression of ZEB1 and ZEB2 on CSC properties in HNC-CD133+ and HNC-CD133 cells in vitro and in vivo. Our findings showed that ZEB1/2 depletion inhibited the cancer stem-like properties and tumor-initiating capabilities of HNC-CD133+ cells. In addition, a high expression of both ZEB1 and ZEB2 predicts poor prognosis in HNC patients.

Section snippets

HNC tissues acquirement and preparation

The study was approved by the institutional review board of Taipei Veterans General Hospital. Resected tissues from HNC patients (Table 1), who gave informed consent for the use of their tissue, were harvested at surgery. Human primary HNC, along with as well as available metastatic lesions were obtained from surgical procedures send to the pathology lab for frozen section diagnosis. Tumor tissues were microscopically screened to have >70% of their areas occupied by tumor cells; The remaining

Up-regulation of ZEB1 and ZEB2 in HNC-derived CD133-positive cells

Recent studies have shown that the expression of CD133 in head and neck cancer results in high tumorigenicity and resistance to conventional therapy.11, 13 To further explore other possible cancer stem-like cell (CSC) characteristics of CD133+ HNC cells, we isolated CD133+ cells from two HNC patient tissue samples using the magnetic bead method (Fig. 1A). The self-renewal ability to form floating spheroid-like bodies (SBs) after serial passages was significantly higher in HNC-CD133+ cells than

Discussion

Epithelial–mesenchymal transition (EMT), a de-differentiation program that converts adherent epithelial cells into individual migratory cells, is critical for embryonic development and the oncogenic progression of tumor cells.28 The EMT process disrupts E-cadherin-mediated cell–cell adhesion during embryonic development and changes the cell phenotype into a less adherent mesenchymal-like cell that can invade the extracellular matrix.29 Extensive studies have revealed that several signaling

Conflict of interest statement

None declared.

Acknowledgments

This work was supported, in part, by Research Grants from Taiwan National Science Council Research Grant (NSC100-2314-B-040-010-MY3, NSC100-2632-B-040-001-MY3, NSC100-2314-B-075-017-MY3, NSC100-2314-B-075-038, NSC100-2314-B-075-031).

References (45)

  • F. Yu et al.

    Let-7 regulates self renewal and tumorigenicity of breast cancer cells

    Cell

    (2007)
  • Y. Shimono et al.

    Downregulation of miRNA-200c links breast cancer stem cells with normal stem cells

    Cell

    (2009)
  • D. Iliopoulos et al.

    Loss of miR-200 inhibition of Suz12 leads to polycomb-mediated repression required for the formation and maintenance of cancer stem cells

    Mol Cell

    (2010)
  • A. Jemal et al.

    Cancer statistics, 2008

    CA: Cancer J Clin

    (2008)
  • J.M. Rosen et al.

    The increasing complexity of the cancer stem cell paradigm

    Science

    (2009)
  • W.L. Lo et al.

    MicroRNA-200c attenuates tumour growth and metastasis of presumptive head and neck squamous cell carcinoma stem cells

    J Pathol

    (2011)
  • P.B. Gupta et al.

    Cancer stem cells: mirage or reality?

    Nat Med

    (2009)
  • M.J. Wu et al.

    Elimination of head and neck cancer initiating cells through targeting glucose regulated protein78 signaling

    Mol Cancer

    (2010)
  • S.K. Singh et al.

    Identification of human brain tumour initiating cells

    Nature

    (2004)
  • F. Zeppernick et al.

    Stem cell marker CD133 affects clinical outcome in glioma patients

    Clin Cancer Res: Off J Am Assoc Cancer Res

    (2008)
  • Y.S. Chen et al.

    CD133/Src axis mediates tumor initiating property and epithelial–mesenchymal transition of head and neck cancer

    PLoS One

    (2011)
  • N.N. Waldron et al.

    Targeting tumor-initiating cancer cells with dCD133KDEL shows impressive tumor reductions in a xenotransplant model of human head and neck cancer

    Mol Cancer Ther

    (2011)
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