Skip to main content

Advertisement

SpringerLink
Log in

Overexpression of Sal-like protein 4 in head and neck cancer: epigenetic effects and clinical correlations

  • Original paper
  • Published:
Cellular Oncology Aims and scope Submit manuscript

Abstract

Background

Sal-like protein 4 (SALL4), an embryonic stem cell factor, has been reported to play an essential role in embryogenesis and oncogenesis. As yet, however, the expression and role of this transcription factor in head and neck squamous cell carcinoma (HNSCC) has not been established.

Methods

We assessed SALL4 mRNA expression in a well-characterised dataset of 230 HNSCC samples (test cohort 110 cases and validation cohort 120 cases). We also transfected HNSCC cells (FaDu and UM-SCC-6) with SALL4 siRNA and assessed its effects on proliferation and expression of specific epigenetic factors in order to uncover the role of SALL4 in HNSCC.

Results

Overexpression of SALL4 was detected in tumour samples of both cohorts. HNSCC cells treated with SALL4 siRNA showed a reduction in growth and a decrease in DNA methyltransferase 3 alpha (DNMT3A) expression. In the patient cohorts, SALL4 overexpression was found to significantly correlate with disease recurrence (p < 0.001) and SALL4 methylation status (p = 0.002). We also found that DNMT3A was significantly upregulated upon SALL4 upregulation (p < 0.001). High expression levels of SALL4 correlated with decreases in disease-free survival (DFS) rates (log-rank test, p < 0.001). Multivariate analysis revealed that SALL4 expression served as an independent prognostic factor for DFS (hazard ratio: 2.566, 95% confidence interval: 1.598–4.121; p < 0.001).

Conclusions

Our findings indicate that SALL4 upregulation correlates with HNSCC tumour aggressiveness and an adverse patient outcome. Our findings also indicate that DNMT3A may synergistically contribute to the regulatory effects of SALL4. Our findings provide insight into SALL4-mediated HNSCC development via epigenetic modulation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

SALL4:

Sal-like protein 4

HNSCC:

head and neck squamous cell carcinoma

DNMT3A:

DNA methyltransferase 3 alpha

DFS:

disease-free survival

ROC:

Receiver operating characteristic

TCGA:

The Cancer Genome Atlas

EGFR:

epidermal growth factor receptor

qRT-PCR:

quantitative reverse transcription PCR

Q-MSP:

quantitative methylation-specific PCR

References

  1. M.R. Migden, D. Rischin, C.D. Schmults, A. Guminski, A. Hauschild, K.D. Lewis, C.H. Chung, L. Hernandez-Aya, A.M. Lim, A.L.S. Chang, G. Rabinowits, A.A. Thai, L.A. Dunn, B.G.M. Hughes, N.I. Khushalani, B. Modi, D. Schadendorf, B. Gao, F. Seebach, S. Li, J. Li, M. Mathias, J. Booth, K. Mohan, E. Stankevich, H.M. Babiker, I. Brana, M. Gil-Martin, J. Homsi, M.L. Johnson, V. Moreno, J. Niu, T.K. Owonikoko, K.P. Papadopoulos, G.D. Yancopoulos, I. Lowy, M.G. Fury, PD-1 Blockade with cemiplimab in advanced cutaneous squamous-cell carcinoma. N Engl J Med 379, 341–351 (2018)

  2. T. Kanazawa, K. Misawa, Y. Misawa, T. Uehara, H. Fukushima, G. Kusaka, M. Maruta, T.E. Carey, G-protein-coupled receptors: Next generation therapeutic targets in head and neck cancer? Toxins (Basel) 7, 2959–2984 (2015)

  3. M. Mikulandra, A. Kobescak, B. Verillaud, P. Busson, T. Matijevic Glavan, Radio-sensitization of head and neck cancer cells by a combination of poly(I:C) and cisplatin through downregulation of survivin and c-IAP2. Cell Oncol 42, 29–40 (2019)

  4. S. Roy, M. Kar, A. Saha, S. Padhi, B. Banerjee, Role of beta-catenin in cisplatin resistance, relapse and prognosis of head and neck squamous cell carcinoma. Cell Oncol 41, 185–200 (2018)

  5. V. Budach, I. Tinhofer, Novel prognostic clinical factors and biomarkers for outcome prediction in head and neck cancer: A systematic review. Lancet Oncol 20, e313–e326 (2019)

    Article  Google Scholar 

  6. J.Y. Hur, H.R. Kim, J.Y. Lee, S. Park, J.A. Hwang, W.S. Kim, S. Yoon, C.M. Choi, J.K. Rho, J.C. Lee, CDK7 inhibition as a promising therapeutic strategy for lung squamous cell carcinomas with a SOX2 amplification. Cell Oncol 42, 449–458 (2019)

  7. P. Boscolo-Rizzo, M.C. Da Mosto, E. Rampazzo, S. Giunco, A. Del Mistro, A. Menegaldo, L. Baboci, M. Mantovani, G. Tirelli, A. De Rossi, Telomeres and telomerase in head and neck squamous cell carcinoma: From pathogenesis to clinical implications. Cancer Metastasis Rev 35, 457–474 (2016)

    Article  CAS  Google Scholar 

  8. K. Misawa, Y. Misawa, T. Kanazawa, D. Mochizuki, A. Imai, S. Endo, T.E. Carey, H. Mineta, Epigenetic inactivation of galanin and GALR1/2 is associated with early recurrence in head and neck cancer. Clin Exp Metastasis 33, 187–195 (2016)

    Article  CAS  Google Scholar 

  9. T. Nakagawa, K. Matsusaka, K. Misawa, S. Ota, K. Takane, M. Fukuyo, B. Rahmutulla, K.I. Shinohara, N. Kunii, D. Sakurai, T. Hanazawa, H. Matsubara, Y. Nakatani, Y. Okamoto, A. Kaneda, Frequent promoter hypermethylation associated with human papillomavirus infection in pharyngeal cancer. Cancer Lett 407, 21–31 (2017)

    Article  CAS  Google Scholar 

  10. J. Kohlhase, R. Schuh, G. Dowe, R.P. Kuhnlein, H. Jackle, B. Schroeder, W. Schulz-Schaeffer, H.A. Kretzschmar, A. Kohler, U. Muller, M. Raab-Vetter, E. Burkhardt, W. Engel, R. Stick, Isolation, characterization, and organ-specific expression of two novel human zinc finger genes related to the Drosophila gene Spalt. Genomics 38, 291–298 (1996)

    Article  CAS  Google Scholar 

  11. L. Yang, L. Liu, H. Gao, J.P. Pinnamaneni, D. Sanagasetti, V.P. Singh, K. Wang, M. Mathison, Q. Zhang, F. Chen, Q. Mo, T. Rosengart, J. Yang, The stem cell factor SALL4 is an essential transcriptional regulator in mixed lineage leukemia-rearranged leukemogenesis. J Hematol Oncol 10, 159 (2017)

    Article  Google Scholar 

  12. H. Tatetsu, N.R. Kong, G. Chong, G. Amabile, D.G. Tenen, L. Chai, SALL4, the missing link between stem cells, development and cancer. Gene 584, 111–119 (2016)

    Article  CAS  Google Scholar 

  13. K. Mei, A. Liu, R.W. Allan, P. Wang, Z. Lane, T.W. Abel, L. Wei, H. Cheng, S. Guo, Y. Peng, D. Rakheja, M. Wang, J. Ma, M.M. Rodriguez, J. Li, D. Cao, Diagnostic utility of SALL4 in primary germ cell tumors of the central nervous system: A study of 77 cases. Mod Pathol 22, 1628–1636 (2009)

    Article  CAS  Google Scholar 

  14. K.J. Yong, C. Gao, J.S. Lim, B. Yan, H. Yang, T. Dimitrov, A. Kawasaki, C.W. Ong, K.F. Wong, S. Lee, S. Ravikumar, S. Srivastava, X. Tian, R.T. Poon, S.T. Fan, J.M. Luk, Y.Y. Dan, M. Salto-Tellez, L. Chai, D.G. Tenen, Oncofetal gene SALL4 in aggressive hepatocellular carcinoma. N Engl J Med 368, 2266–2276 (2013)

    Article  CAS  Google Scholar 

  15. J. Yang, T.R. Corsello, Y. Ma, Stem cell gene SALL4 suppresses transcription through recruitment of DNA methyltransferases. J Biol Chem 287, 1996–2005 (2012)

    Article  CAS  Google Scholar 

  16. J. Xiong, Z. Zhang, J. Chen, H. Huang, Y. Xu, X. Ding, Y. Zheng, R. Nishinakamura, G.L. Xu, H. Wang, S. Chen, S. Gao, B. Zhu, Cooperative action between SALL4A and TET proteins in stepwise oxidation of 5-Methylcytosine. Mol Cell 64, 913–925 (2016)

    Article  CAS  Google Scholar 

  17. K. Misawa, D. Mochizuki, A. Imai, Y. Misawa, S. Endo, M. Mima, H. Kawasaki, T.E. Carey, T. Kanazawa, Epigenetic silencing of SALL3 is an independent predictor of poor survival in head and neck cancer. Clin Epigenetics 9, 64 (2017)

    Article  Google Scholar 

  18. K. Misawa, Y. Misawa, A. Imai, D. Mochizuki, S. Endo, M. Mima, R. Ishikawa, H. Kawasaki, T. Yamatodani, T. Kanazawa, Epigenetic modification of SALL1 as a novel biomarker for the prognosis of early stage head and neck cancer. J Cancer 9, 941–949 (2018)

    Article  Google Scholar 

  19. A. Imai, D. Mochizuki, Y. Misawa, T. Nakagawa, S. Endo, M. Mima, S. Yamada, H. Kawasaki, T. Kanazawa, K. Misawa, SALL2 is a novel prognostic methylation marker in patients with oral squamous carcinomas: Associations with SALL1 and SALL3 methylation status. DNA Cell Biol 38, 678–687 (2019)

  20. D. Mochizuki, Y. Misawa, H. Kawasaki, A. Imai, S. Endo, M. Mima, S. Yamada, T. Nakagawa, T. Kanazawa, K. Misawa, Aberrant epigenetic regulation in head and neck Cancer due to distinct EZH2 overexpression and DNA Hypermethylation. Int J Mol Sci 19 (2018)

  21. K. Misawa, D. Mochizuki, A. Imai, S. Endo, M. Mima, Y. Misawa, T. Kanazawa, T.E. Carey, H. Mineta, Prognostic value of aberrant promoter hypermethylation of tumor-related genes in early-stage head and neck cancer. Oncotarget 7, 26087–26098 (2016)

    Article  Google Scholar 

  22. W.Y. Huang, S.D. Hsu, H.Y. Huang, Y.M. Sun, C.H. Chou, S.L. Weng, H.D. Huang, MethHC: A database of DNA methylation and gene expression in human cancer. Nucleic Acids Res 43, D856–D861 (2015)

    Article  CAS  Google Scholar 

  23. J. Itou, W. Li, S. Ito, S. Tanaka, Y. Matsumoto, F. Sato, M. Toi, Sal-like 4 protein levels in breast cancer cells are post-translationally down-regulated by tripartite motif-containing 21. J Biol Chem 293, 6556–6564 (2018)

    Article  CAS  Google Scholar 

  24. D. Cao, P.A. Humphrey, R.W. Allan, SALL4 is a novel sensitive and specific marker for metastatic germ cell tumors, with particular utility in detection of metastatic yolk sac tumors. Cancer 115, 2640–2651 (2009)

    Article  CAS  Google Scholar 

  25. C. Sun, P. Lan, Q. Han, M. Huang, Z. Zhang, G. Xu, J. Song, J. Wang, H. Wei, J. Zhang, R. Sun, C. Zhang, Z. Tian, Oncofetal gene SALL4 reactivation by hepatitis B virus counteracts miR-200c in PD-L1-induced T cell exhaustion. Nat Commun 9, 1241 (2018)

    Article  Google Scholar 

  26. W. Du, L. Ni, B. Liu, Y. Wei, Y. Lv, S. Qiang, J. Dong, X. Liu, Upregulation of SALL4 by EGFR activation regulates the stemness of CD44-positive lung cancer. Oncogenesis 7, 36 (2018)

    Article  Google Scholar 

  27. J.U. Marquardt, S.S. Thorgeirsson, Sall4 in "stemness"-driven hepatocarcinogenesis. N Engl J Med 368, 2316–2318 (2013)

    Article  CAS  Google Scholar 

  28. S.S. Zeng, T. Yamashita, M. Kondo, K. Nio, T. Hayashi, Y. Hara, Y. Nomura, M. Yoshida, T. Hayashi, N. Oishi, H. Ikeda, M. Honda, S. Kaneko, The transcription factor SALL4 regulates stemness of EpCAM-positive hepatocellular carcinoma. J Hepatol 60, 127–134 (2014)

    Article  CAS  Google Scholar 

  29. L. Vilorio-Marques, V. Martin, C. Diez-Tascon, M.F. Gonzalez-Sevilla, T. Fernandez-Villa, E. Honrado, V. Davila-Batista, A.J. Molina, The role of EZH2 in overall survival of colorectal cancer: A meta-analysis. Sci Rep 7, 13806 (2017)

    Article  Google Scholar 

  30. J. Itou, Y. Matsumoto, K. Yoshikawa, M. Toi, Sal-like 4 (SALL4) suppresses CDH1 expression and maintains cell dispersion in basal-like breast cancer. FEBS Lett 587, 3115–3121 (2013)

    Article  CAS  Google Scholar 

  31. J. He, M. Zhou, X. Chen, D. Yue, L. Yang, G. Qin, Z. Zhang, Q. Gao, D. Wang, C. Zhang, L. Huang, L. Wang, B. Zhang, J. Yu, Y. Zhang, Inhibition of SALL4 reduces tumorigenicity involving epithelial-mesenchymal transition via Wnt/beta-catenin pathway in esophageal squamous cell carcinoma. J Exp Clin Cancer Res 35, 98 (2016)

    Article  Google Scholar 

  32. K. Nimura, K. Ura, H. Shiratori, M. Ikawa, M. Okabe, R.J. Schwartz, Y. Kaneda, A histone H3 lysine 36 trimethyltransferase links Nkx2-5 to wolf-Hirschhorn syndrome. Nature 460, 287–291 (2009)

    Article  CAS  Google Scholar 

  33. J. Yang, SALL4 as a transcriptional and epigenetic regulator in normal and leukemic hematopoiesis. Biomark Res 6, 1 (2018)

    Article  CAS  Google Scholar 

  34. T. Baubec, D.F. Colombo, C. Wirbelauer, J. Schmidt, L. Burger, A.R. Krebs, A. Akalin, D. Schubeler, Genomic profiling of DNA methyltransferases reveals a role for DNMT3B in genic methylation. Nature 520, 243–247 (2015)

    Article  CAS  Google Scholar 

  35. J. Lu, H.W. Jeong, N. Kong, Y. Yang, J. Carroll, H.R. Luo, L.E. Silberstein, Yupoma, L. Chai, Stem cell factor SALL4 represses the transcriptions of PTEN and SALL1 through an epigenetic repressor complex. PLoS One 4, e5577 (2009)

    Article  Google Scholar 

  36. A. Li, Y. Jiao, K.J. Yong, F. Wang, C. Gao, B. Yan, S. Srivastava, G.S. Lim, P. Tang, H. Yang, D.G. Tenen, L. Chai, SALL4 is a new target in endometrial cancer. Oncogene 34, 63–72 (2015)

    Article  CAS  Google Scholar 

  37. X. Zhang, X. Yuan, W. Zhu, H. Qian, W. Xu, SALL4: An emerging cancer biomarker and target. Cancer Lett 357, 55–62 (2015)

    Article  CAS  Google Scholar 

  38. Y. Ma, W. Cui, J. Yang, J. Qu, C. Di, H.M. Amin, R. Lai, J. Ritz, D.S. Krause, L. Chai, SALL4, a novel oncogene, is constitutively expressed in human acute myeloid leukemia (AML) and induces AML in transgenic mice. Blood 108, 2726–2735 (2006)

    Article  CAS  Google Scholar 

  39. X. Yue, L. Xiao, Y. Yang, W. Liu, K. Zhang, G. Shi, H. Zhou, J. Geng, X. Ning, J. Wu, Q. Zhang, High cytoplasmic expression of SALL4 predicts a malignant phenotype and poor prognosis of breast invasive ductal carcinoma. Neoplasma 62, 980–988 (2015)

    Article  CAS  Google Scholar 

  40. L. Zhang, Y. Yan, Y. Jiang, Y. Cui, Y. Zou, J. Qian, C. Luo, Y. Lu, X. Wu, The expression of SALL4 in patients with gliomas: High level of SALL4 expression is correlated with poor outcome. J Neuro-Oncol 121, 261–268 (2015)

    Article  CAS  Google Scholar 

  41. S. Onder, O.C. Taskin, F. Sen, S. Topuz, S. Kucucuk, H. Sozen, R. Ilhan, S. Tuzlali, E. Yavuz, High expression of SALL4 and fascin, and loss of E-cadherin expression in undifferentiated/dedifferentiated carcinomas of the endometrium: An immunohistochemical and clinicopathologic study. Medicine (Baltimore) 96, e6248 (2017)

  42. N. Yanagihara, D. Kobayashi, K. Kuribayashi, M. Tanaka, T. Hasegawa, N. Watanabe, Significance of SALL4 as a drugresistant factor in lung cancer. Int J Oncol 46, 1527–1534 (2015)

    Article  CAS  Google Scholar 

  43. M. Miettinen, Z. Wang, P.A. McCue, M. Sarlomo-Rikala, J. Rys, W. Biernat, J. Lasota, Y.S. Lee, SALL4 expression in germ cell and non-germ cell tumors: A systematic immunohistochemical study of 3215 cases. Am J Surg Pathol 38, 410–420 (2014)

    Article  Google Scholar 

  44. S. Zhou, R. Venkatramani, E. Gomulia, N. Shillingford, L. Wang, The diagnostic and prognostic value of SALL4 in hepatoblastoma. Histopathology 69, 822–830 (2016)

    Article  Google Scholar 

  45. X. Zhang, P. Zhang, M. Shao, X. Zang, J. Zhang, F. Mao, H. Qian, W. Xu, SALL4 activates TGF-beta/SMAD signaling pathway to induce EMT and promote gastric cancer metastasis. Cancer Manag Res 10, 4459–4470 (2018)

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Ms. Yuko Mohri for her excellent technical support and Editage (www.editage.jp) for English language editing.

Funding

This study was funded by a Grant-in-Aid for Scientific Research (No. 17 K11380, No. 17 K16903, 17 K16904, 19 K09866, 19 K09906 and 19 K18728) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Author information

Authors and Affiliations

  1. Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine, 1-20-1 Handayama, Shizuoka, 431-3192, Japan

    Kiyoshi Misawa, Yuki Misawa, Masato Mima, Satoshi Yamada, Atsushi Imai, Daiki Mochizuki, Shiori Endo & Hiroyuki Mineta

  2. Department of Molecular Oncology, Chiba University, Chiba, Japan

    Masato Mima, Takuya Nakagawa & Tomoya Kurokawa

  3. Preeminent Medical Photonics Education & Research Center Institute for NanoSuit Research, Hamamatsu University School of Medicine, Hamamatsu, Japan

    Satoshi Yamada & Hideya Kawasaki

  4. Department of Otolaryngology-Head and Neck Surgery, University of Michigan Health System, Ann Arbor, MI, USA

    John Chadwick. Brenner

Authors
  1. Kiyoshi Misawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yuki Misawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Masato Mima
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Satoshi Yamada
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Atsushi Imai
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Daiki Mochizuki
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Takuya Nakagawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Tomoya Kurokawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Shiori Endo
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Hideya Kawasaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. John Chadwick. Brenner
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Hiroyuki Mineta
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

KM and YM conceived the study. KM, YM and KH designed the experiments. MM, YS, SI, DM, TK, TK, SE and MM analysed the data and prepared the figures and tables. All authors participated in writing the manuscript, reviewed its drafts, approved its final version and agreed with its submission.

Corresponding author

Correspondence to Kiyoshi Misawa.

Ethics declarations

Conflict of interest

None to declare.

Ethics approval and consent to participate

The research methodology employed in this study was approved by The Institutional Review Board of the Hamamatsu University School of Medicine. All study subjects provided written informed consent.

Consent for publication

Consent for publication was obtained from all patients.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Supplementary Fig. S1

Kaplan–Meier survival curves for patients with (A) oral cancer (n = 76; p = 0.026), (B) hypopharyngeal cancer (n = 54; p = 0.420), (C) laryngeal cancer (n = 51; p = 0.173), and (D) oropharyngeal cancer (n = 35; p = 0.111). * p < 0.05. (PNG 89 kb)

High Resolution (EPS 1083 kb)

Supplementary Fig. S2

Data regarding SALL4 (A), DNMT3A (B), and DNMT3B (C) mRNA expression in head and neck squamous cell carcinoma were obtained from TCGA (https://tcga-data.nci.nih.gov/tcga/) and MethHC (http://methhc.mbc.nctu.edu.tw/php/index.php). * p < 0.05, ** p < 0.01. (PNG 94 kb)

High Resolution (EPS 1594 kb)

Supplementary Fig. S3

Data regarding SALL4, CDH1 (A), COL1A2 (B) and CDH13 (C) mRNA expression in head and neck squamous cell carcinoma were obtained from TCGA (https://tcga-data.nci.nih.gov/tcga/) and MethHC (http://methhc.mbc.nctu.edu.tw/php/index.php). * p < 0.05. (PNG 109 kb)

High Resolution (EPS 1895 kb)

Supplementary Table S1

(DOCX 20 kb)

Supplementary Table S2

(DOCX 19 kb)

Supplementary Table S3

(DOCX 20 kb)

Supplementary Table S4

(DOCX 18 kb)

Supplementary Table S5

(DOCX 21 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Misawa, K., Misawa, Y., Mima, M. et al. Overexpression of Sal-like protein 4 in head and neck cancer: epigenetic effects and clinical correlations. Cell Oncol. 43, 631–641 (2020). https://doi.org/10.1007/s13402-020-00509-5

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13402-020-00509-5

Keywords

Search

Navigation