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EGFR Nuclear Import in Gallbladder Carcinoma: Nuclear Phosphorylated EGFR Upregulates iNOS Expression and Confers Independent Prognostic Impact

  • Hepatobiliary Tumors
  • Published:
Annals of Surgical Oncology Aims and scope Submit manuscript

Abstract

Background

The understanding of epidermal growth factor receptor (EGFR) deregulation in carcinogenesis remains incomplete. We investigated the implications of EGFR gene status and EGFR nuclear translocation in gallbladder carcinoma (GBCA).

Methods

Subcellular localization of EGFR and phosphorylated EGFR (pEGFR) was analyzed by fractional immunoblotting and confocal immunofluorescence in GBCA cell lines. pEGFR binding to iNOS promoter was assessed by chromatin immunoprecipitation with iNOS promoter activity evaluated by luciferase assay. EGFR, pEGFR, and iNOS were immunohistochemically assessable for localization and level in the training set of 104 GBCAs on tissue microarrays, with 76 cases analyzed for EGFR gene by chromogenic in situ hybridization (CISH) and mutant-enriched PCR targeting exons 19 and 21. The prognostic impact of nuclear pEGFR (N-pEGFR) immunoexpression was reaffirmed on whole sections of 58 GBCAs in the test set.

Results

Nuclear expression of EGFR and pEGFR was substantiated in vitro with augmented activity of iNOS promoter elicited by pEGFR binding upon EGF treatment. Despite no mutation, EGFR amplification, identified in 11 cases (15%) by CISH, strongly correlated with cytoplasmic EGFR expression (P < 0.001) but not with disease-specific survival (DSS). Immunoexpression of nuclear EGFR (N-EGFR), cytoplasmic pEGFR, and N-pEGFR was strongly related to that of iNOS (all ≤0.005). N-pEGFR independently predicted worse DSS in both training (P = 0.0468, HR = 2.024) and test sets (P = 0.0223, HR = 5.573).

Conclusions

N-EGFR and N-pEGFR express in GBCA, conferring clinical aggressiveness partly through iNOS transactivation. Lacking response-predicting mutation, EGFR gene status, albeit amplified in 15% of GBCA, is neither related to nuclear EGFR translocation nor prognostically useful.

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Acknowledgment

This study was supported in part by grants from National Science Council, Taiwan (NSC96-2321-B-182A-001-MY2), Chang Gung Memorial Hospital (CMRPG870753), Chi-Mei Medical Center (CMFHR9935), and Department of Health, Taiwan (DOH99-TD-C-111-004). The authors are grateful to genomic core laboratory (CZRPG880253) and tissue bank (CMRPG870461) of Chang Gung Memorial Hospital-Kaohsiung Medical Center. Immense gratitude is given to Dr. Shiu-Feng F. Huang and Dr. Marc Ladanyi for providing critical technical assistance in EGFR chromogenic in situ hybridization.

Conflicts of interest

The authors have no potential conflicts of interest.

Author information

Authors and Affiliations

  1. Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan

    Chien-Feng Li MD & Ching-Cherng Tzeng MD

  2. Institute of Biomedical Science, National Sun Yat-Sen University, Kaohsiung, Taiwan

    Chien-Feng Li MD & Yow-Ling Shiue PhD

  3. National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan

    Chien-Feng Li MD, Patrick Yu-Wei Chu MS & Li-Tzong Chen MD, PhD

  4. Department of Biotechnology, Southern Taiwan University, Tainan, Taiwan

    Chien-Feng Li MD

  5. Department of Radiation Oncology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan

    Fu-Ming Fang MD, PhD

  6. Institute of Biosignal Transduction, National Cheng Kung University, Tainan, Taiwan

    Ju-Ming Wang PhD, Yu-Hui Wang MS & Wen-Ling Wang PhD

  7. Department of Pathology, Changhua Christian Hospital, Changhua, Taiwan

    Hui-Chun Tai MD

  8. Department of Pathology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan

    Yu-Ching Wei MD, Yuan-Ting Lee MS, Yu-Hui Wang MS, Shih-Chen Yu BS & Hsuan-Ying Huang MD

  9. Department of Medical Oncology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan

    Shau-Hsuan Li MD

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  1. Chien-Feng Li MD
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  2. Fu-Ming Fang MD, PhD
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  14. Li-Tzong Chen MD, PhD
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  15. Hsuan-Ying Huang MD
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Corresponding author

Correspondence to Hsuan-Ying Huang MD.

Additional information

Chien-Feng Li, Fu-Ming Fang, and Ju-Ming Wang contributed equally to this work.

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Li, CF., Fang, FM., Wang, JM. et al. EGFR Nuclear Import in Gallbladder Carcinoma: Nuclear Phosphorylated EGFR Upregulates iNOS Expression and Confers Independent Prognostic Impact. Ann Surg Oncol 19, 443–454 (2012). https://doi.org/10.1245/s10434-011-1942-6

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  • DOI: https://doi.org/10.1245/s10434-011-1942-6

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