Elsevier

Journal of Proteomics

Volume 91, 8 October 2013, Pages 453-465
Journal of Proteomics

iTRAQ-based quantitative proteomics reveals myoferlin as a novel prognostic predictor in pancreatic adenocarcinoma

https://doi.org/10.1016/j.jprot.2013.06.032Get rights and content

Highlights

  • Identify the proteins involved in histological differentiation of pancreatic cancer.

  • Two sets of six different isobaric tags were used for the four independent groups.

  • Myoferlin is an independent prognostic factor for survival of PC patients.

  • Knockdown of MYOF alleviated malignant phenotypes of PC cell lines.

Abstract

Histological differentiation is a major pathological parameter associated with poor prognosis in patients with pancreatic adenocarcinoma (PAC) and the molecular signature underlying PAC differentiation may involve key proteins potentially affecting the malignant characters of PAC. We aimed to identify the proteins which could be implicated in PAC prognosis. We used isobaric tags for relative and absolute quantitation (iTRAQ) coupled with two-dimensional liquid chromatography–tandem mass spectrometry to compare protein expression in PAC tissues with different degrees of histological differentiation. A total of 1623 proteins were repeatedly identified by performing the iTRAQ-based experiments twice. Of these, 15 proteins were differentially expressed according to our defined criteria. Myoferlin (MYOF) was selected to validate the proteomic results by western blotting. Immunohistochemistry in a further 154 PAC cases revealed that myoferlin significantly correlated with the degree of histological differentiation (P = 0.004), and univariate and multivariate analyses indicated that MYOF is an independent prognostic factor for survival (hazard ratio, 1.540; 95% confidence interval, 1.061–2.234; P = 0.023) of patients with PAC after curative surgery. RNA interference-mediated knockdown of MYOF alleviated malignant phenotypes of both primary and metastatic PAC cell lines in vitro and in vivo. Thus, ITRAQ-based quantitative proteomics revealed the prognostic value of MYOF in PAC.

Biological significance

Our results provide the possibility of novel strategies for pancreatic adenocarcinoma management.

Introduction

Pancreatic adenocarcinoma (PAC) is a lethal malignancy with an overall 5-year survival rate of only approximately 5% [1]. In 2008, PAC was responsible for an estimated 268,800 deaths worldwide [2]. Thus, novel diagnostic and therapeutic modalities have long been required to improve the clinical outcome of patients with PAC. Tumor grade is an important prognostic variable of survival in PAC; patients with poorly-differentiated tumors tend to have worse prognosis than those with well-differentiated tumors [3]. Therefore, the molecular background of tumor grade may be implicated in prognostic biomarker candidates, which may lead to novel strategies for PAC management.

Isobaric tags for relative and absolute quantitation (iTRAQ) combined with two-dimensional liquid chromatography–tandem mass spectrometry (2D LC–MS/MS) is one of most powerful methodologies in quantitative proteomics [4], [5], [6]. The iTRAQ reagent labels the primary amines on the peptides and thus can allow the tagging of most tryptic peptides, theoretically. The multiplexing ability afforded by the iTRAQ reagents, which are available in four to eight different tags, suited the design of our present study.

In our study, we revealed that overexpression of myoferlin (MYOF) was correlated with high tumor grade in PAC by iTRAQ labeling combined with 2D LC–MS/MS. Myoferlin has been identified as a membrane-associated protein important for muscle development and regeneration [7], [8]. Immunohistochemical evidence available from the Human Protein Atlas [9] suggests that myoferlin is strongly expressed in several cancer types including colorectal, breast, ovarian, cervical, endometrial, thyroid, stomach, pancreatic, and liver cancers [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20]. Eisenberg et al. [21] used a combination of mathematical modeling and experimental validation to uncover myoferlin as a cancer protein, which could be involved in regulating cancer cell invasion. Li et al. [22] demonstrated that myoferlin depletion in breast cancer cells promotes mesenchymal to epithelial shape change and stalls invasion. Recently, Leung et al. [23] suggested that interfering with normal myoferlin expression and/or membrane repair and remodeling may provide therapeutically relevant antiproliferative effects. However, the function of myoferlin in PAC has not been reported. We hypothesized that PAC patients with overexpression of myoferlin could have worse prognosis. Therefore, we examined an additional 154 patients with PAC who underwent initial surgical resection to evaluate the correlation between the two variables. And we further explored if RNA interference-mediated knockdown of MYOF alleviated malignant phenotypes of PAC cell lines in vitro and in vivo.

Section snippets

Patients and tissue samples

A total of 60 surgically resected fresh tissues (~ 0.5 cm3) were obtained from 40 PAC patients who underwent initial pancreatic resection at the Zhongshan Hospital between June 2010 and January 2012. Fresh tissues were stored at − 80 °C until use. The tissue samples were divided into three categories: low-grade PAC (n = 20), high-grade PAC (n = 20), and nontumor pancreatic tissues (≥ 3 cm away from the pancreatic tumors) adjacent to tumors (n = 20). For PAC, histologic grade is based on the extent of

Protein identification

A total of 2844 proteins were identified by the first iTRAQ-based experiment (Supplementary Table S4), while a total of 2588 proteins by the second (Supplementary Table S5). And then 1623 proteins were repeatedly identified and quantified by performing the iTRAQ-based experiments twice. To identify the differentially expressed proteins in the high-grade PAC tissues, protein profiles between two types of tissues (high-grade PAC tissues vs. low-grade PAC tissues, or adjacent nontumor tissues)

Discussion

In the present study, iTRAQ labeling combined with 2D LC–MS/MS was used to identify differentially expressed proteins in high-grade PAC tissues, compared with low-grade PAC tissues and with adjacent nontumor tissues. The traditional iTRAQ technique was followed, except with some modifications in the study design: the 60 surgically resected tissues obtained from 40 PAC patients (20 patients with low-grade tumor, and 20 patients with high-grade tumor) were divided into four groups (I, II, III,

New findings

  • 1.

    Our results revealed that myoferlin significantly correlated with the degree of histological differentiation and that it is an independent prognostic factor for survival of patients with pancreatic adenocarcinoma after curative surgery.

  • 2.

    RNA interference–mediated knockdown of MYOF alleviated malignant phenotypes of both primary and metastatic PAC cell lines in vitro and in vivo.

Conflict of interest

The authors indicated no potential conflicts of interest.

The authors have disclosed to study participants that they have no potential investigator conflicts of interest.

Contributorship

Conception and design: Xiao-Lin Wang, Peng-yuan Yang

Financial support: Xiao-Lin Wang, Peng-yuan Yang

Administrative support: Xiao-Lin Wang, Peng-yuan Yang

Provision of study materials or patients: Xiao-Lin Wang, Wen-Hui Lou, Da-Yong Jin

Collection and assembly of data: Wan-Sheng Wang, Ling-Xiao Liu, Wei Zhang, Guo-Ping Li, Yi Chen, Chang-Yu Li, Xiao-Hui Liu

Data analysis and interpretation: Wan-Sheng Wang, Xiao-Lin Wang

Manuscript writing: All authors

Final approval of manuscript: All authors

Funding

Supported by the Program of Advanced Technology of Hospitals in Shanghai Grant No. SHDC12010120.

References (32)

  • A. Jemal et al.

    Global cancer statistics

    CA Cancer J Clin

    (2011)
  • N. Wasif et al.

    Impact of tumor grade on prognosis in pancreatic cancer: should we include grade in AJCC staging?

    Ann Surg Oncol

    (2010)
  • L.R. Zieske

    A perspective on the use of iTRAQ reagent technology for protein complex and profiling studies

    J Exp Bot

    (2006)
  • K.R. Doherty et al.

    Normal myoblast fusion requires myoferlin

    Development

    (2005)
  • F. Pontén et al.

    The human protein atlas—a tool for pathology

    J Pathol

    (2008)
  • S. Amatschek et al.

    Tissue-wide expression profiling using cDNA subtraction and microarrays to identify tumor-specific genes

    Cancer Res

    (2004)
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    Xiao-Lin Wang, Da-Yong Jin, Wen-Hui Lou, and Peng-Yuan Yang contributed equally to this work.

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