Elsevier

Human Pathology

Volume 40, Issue 1, January 2009, Pages 83-91
Human Pathology

Original contribution
Overexpression and altered subcellular localization of autophagy-related 16-like 1 in human oral squamous-cell carcinoma: correlation with lymphovascular invasion and lymph-node metastasis

https://doi.org/10.1016/j.humpath.2008.06.018Get rights and content

Summary

Autophagy is a dynamic process of subcellular degradation, which has recently sparked great interest because it is involved in various developmental processes and various diseases including cancer. Autophagy-related 16-like 1 is a component of a large protein complex essential for autophagosome formation. We previously applied proteomic methods to characterize differentially expressed proteins in oral squamous cell carcinoma cells and detected significantly high expression levels of autophagy-related 16-like 1 in oral squamous cell carcinoma–derived cell lines compared to human normal oral keratinocytes. In the current study, to further determine the potential involvement of autophagy-related 16-like 1 in oral squamous cell carcinoma, we evaluated the state of autophagy-related 16-like 1 protein expression in human oral premalignant lesions and primary oral squamous cell carcinomas, and correlated the results with clinicopathologic variables. Autophagy-related 16-like 1 immunoreaction was predominant in a variety of subcellular components of oral squamous cell carcinoma tissues, including the cytoplasm and plasma membrane of malignant cells (45% and 39%, respectively) and peritumoral and intratumoral stroma (52%), whereas all of the components in normal tissues had no or faint autophagy-related 16-like 1 expression. In addition, high stromal expression of autophagy-related 16-like 1 was associated significantly with lymphovascular invasion of tumor cells (P = .037) and positive lymph node status (P = .015). Furthermore, cytoplasmic and plasma membranous autophagy-related 16-like 1 were also expressed in abundance in the oral premalignant lesion cells (74% and 32%, respectively). Our finding suggests that dysregulation of autophagy-related 16-like 1 protein expression is a frequent and early event during oral carcinogenesis and could affect the malignant behavior of oral squamous cell carcinoma cells.

Introduction

Oral squamous cell carcinoma (OSCC) is a major cause of morbidity and mortality globally, accounting for 275000 new cases and more than 120000 deaths annually [1]. Despite therapeutic and diagnostic advances, patients are often diagnosed at advanced stages and mortality rates are still increasing [2]. This highlights the need for continued efforts to uncover suitable biomarkers for early disease diagnosis and to understand disease pathogenesis as a first step toward improving treatment. We recently developed a strategy of using proteomic technologies to search for significant molecular biomarker characteristics of oral carcinogenesis [3]. Among the proteins identified, autophagy-related 16-like 1 (ATG16L1) expression was found to be upregulated in OSCC-derived cell lines compared to human normal oral keratinocytes using a fluorescent 2-dimensional differential in-gel electrophoresis system and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

ATG16L1, also known as Atg16 or Atg16L, is a member of the autophagy-related protein family, which has an essential role in autophagy. Autophagy mediates bulk degradation of the cytoplasmic component in lysosomes and vacuoles [4], and is implicated in numerous biologic processes, including the anti-aging mechanism [5], and in the survival response during nutrient depletion, or in the absence of growth factors, or both [6]. Autophagy involves formation of a double-membrane vesicle, which encapsulates cytoplasm and organelles and then fuses with lysosomes, thus degrading the vesicle contents [7]. The formation of the autophagosome depends on autophagy-related proteins [8].

The ATG16L1 gene was originally identified in yeast [9]. In mammalians, the mouse homologue of ATG16L1, Atg16L, was identified initially as a functional counterpart of yeast Atg16, which binds to the Atg12-Atg5 conjugate and forms a multimeric complex crucial to autophagosome formation [10]. Human ATG16L1 was first identified in human fetal brain cDNA library, which shows 93% similarity to mouse orthologue Atg16L [11]. A recent study showed that functional knockdown of the gene using RNA-silencing methods in a human HeLa cell line abrogates the autophagy of Salmonella typhimurium[12], suggesting that human ATG16L1 is also indispensable for autophagic function.

Although many recent publications have highlighted the role of autophagy in the regulation of cancer development and progression [13], [14], ATG16L1 has not been characterized previously in the human tumors including OSCC, for which nothing is known in the context of human carcinogenesis. The purpose of the current study was to determine ATG16L1 protein expression in a series of human primary OSCCs and oral premalignant lesions (OPLs) and correlate the expression with the clinical relevance in patients with OSCC.

Section snippets

Tissue specimens

Thirty-three pairs of typical keratinizing-type OSCC samples (not including verrucous or other variant types) and corresponding normal oral epithelium tissues were retrieved nonselectively from our hospital files and pathology database from 1999 to 2006. All patients (23 men, 10 women; mean age, 63.0+13.4 years; range, 27-83 years) provided informed consent according to the protocol that was reviewed and approved by the institutional review board of Chiba University before any procedures were

Expression and subcellular localization of ATG16l1 in OPLs and OSCCs

Immunohistochemical staining was performed using a series of OSCC specimens obtained intraoperatively, including 33 OSCCs with corresponding normal tissues and 31 OPLs that were diagnosed histopathologically as leukoplakia with epithelial dysplasia. Considering that evidence showed that the malignant transformation rate of oral leukoplakia with dysplasia was apparently higher than that of oral leukoplakia without dysplasia [18], patients with advanced OPLs, defined as leukoplakia exhibiting

Discussion

Of particular interest in the current results is that, although it is well known that most mammalian Atg16L is normally present in the cytoplasm as a complex with the Atg12-Atg5 conjugate [10], ATG16L1 protein subcellular distribution at a high expression level was observed in the cytoplasm and the plasma membrane of OSCC cells, whereas no or faint protein expression was detected in either compartment in normal oral epithelium cells. Furthermore, strong ATG16L1 immunoreaction was also

Acknowledgments

We thank Lynda C. Charters for editing this manuscript.

References (35)

  • MorettiL. et al.

    Autophagy signaling in cancer and its potential as novel target to improve anticancer therapy

    Drug Resist Updat

    (2007)
  • SudboJ. et al.

    The evolution of predictive oncology and molecular-based therapy for oral cancer prevention

    Int J Cancer

    (2005)
  • KoikeH. et al.

    Identification of differentially expressed proteins in oral squamous cell carcinoma using a global proteomic approach

    Int J Oncol

    (2005)
  • CodognoP. et al.

    Autophagy and signaling: their role in cell survival and cell death

    Cell Death Differ

    (2005)
  • MizushimaN. et al.

    Autophagosome formation in mammalian cells

    Cell Struct Funct

    (2002)
  • MizushimaN. et al.

    Apg16p is required for the function of the Apg12p-Apg5p conjugate in the yeast autophagy pathway

    EMBO J

    (1999)
  • MizushimaN. et al.

    Mouse Apg16L, a novel WD-repeat protein, targets to the autophagic isolation membrane with the Apg12-Apg5 conjugate

    J Cell Sci

    (2003)
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    This study was partly supported by a Grant-in-Aid Scientific Research (No. 16209059) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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