Sineoculis homeobox homolog 1 protein overexpression as an independent biomarker for pancreatic ductal adenocarcinoma

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Abstract

Sineoculis homeobox homolog 1 (SIX1) is a member of the SIX gene family. It is highly expressed in cancers derived from tissues that play a fundamental role during embryogenesis. Recent studies suggest that inappropriate expression of SIX1 can both initiate tumorigenesis and promote metastasis. To investigate the clinicopathological significance of SIX1 expression in pancreatic ductal adenocarcinoma (PDAC), and to further identify its role as a potential biomarker and therapeutic target in PDAC, 103 PDAC tissue samples and 45 normal pancreatic tissue samples were immunohistochemically stained for SIX1 protein. The localization of SIX1 protein was detected in Panc-1 cancer cells using immunofluorescence staining. Correlations between SIX1 overexpression and the clinicopathological features of pancreatic cancer were evaluated using Chi-square (χ2) tests, differences in survival curves were analyzed using log-rank tests, and multivariate survival analysis was performed using the Cox proportional hazard regression model. In results, SIX1 protein showed mainly cytoplasmic/perinuclear staining pattern in PDAC with immunohistochemistry. The strongly positive rate of SIX1 protein was 60.2% (62/103) in PDAC, which was significantly higher than normal pancreatic tissue (6.7%, 3/45). SIX1 overexpression was positively correlated with tumor size, TNM stage, lymph node metastasis, and grade of PDAC (P < 0.001). SIX1 high expression levels influenced overall survival rates in G1, G2, stage I–II and stage III–IV groups of PDAC; and high expression levels had significantly lower overall survival rates than SIX1 low expression levels. In conclusion, SIX1 emerged as a significant independent prognostic factor in PDAC. SIX1 overexpression appears to be associated with PDAC, and may be a potential biomarker for early diagnosis and prognostic evaluation of PDAC.

Introduction

Pancreatic cancer is the fourth leading cause of cancer-related death, with a 5-year survival rate of 6% (Jemal et al., 2010). The vast majority of solid pancreatic tumors are pancreatic ductal adenocarcinomas (PDAC). The extremely aggressive tumor growth rate and the high incidence of metastasis are characteristics of PDAC. Most patients are in an advanced or metastatic condition at the time of diagnosis, which results in a poor prognosis (Chatterjee et al., 2012, Li et al., 2004). Recurrence remains the main cause of cancer-related death after curative resection in a substantial proportion of patients with PDAC (Lee et al., 2012). Therefore, the identification of a reliable biomarker for predicting recurrence and for identifying tumors is of great interest not only for understanding the molecular and cellular processes involved, but also for searching for possible new therapeutic molecular targets.

Sineoculis homeobox homolog 1 (SIX1), one of the SIX family members, which was first identified and characterized as a mammalian homolog of the Drosophila sineoculis (so) gene (Oliver et al., 1995), is a homeodomain transcription factor and has been implicated in tumor onset (Coletta et al., 2008), progression (Coletta et al., 2004) and embryogenesis (Hendry et al., 2013, Li et al., 2002). During normal development, SIX1 stimulates the proliferation and survival of progenitor cells (Ikeda et al., 2010, McCoy et al., 2009). The loss of function of SIX1 results in a reduction in size or the absence of various organs, because of a decrease in proliferation and an increase in apoptosis (Li et al., 2003, Ozaki et al., 2004, Zheng et al., 2003). Recent studies showed that SIX1 overexpression correlates with a poor prognosis in numerous cancers including ovarian cancer, hepatocellular carcinoma and cervical cancers (Behbakht et al., 2007, Ng et al., 2006, Tan et al., 2011). The overexpression of SIX1 protein likely induces transformation through its ability to increase proliferation (Coletta et al., 2004), survival (Behbakht et al., 2007), and genomic instability (Coletta et al., 2008). A study by Iwanaga (Iwanaga et al., 2012) reported for the first time that SIX1 expression correlated with poor prognosis in luminal breast cancers, especially in the aggressive luminal B subtype. Recently, Li (Li et al., 2013) reported that SIX1 mRNA is overexpressed in pancreatic cancer and correlated with the advanced tumor stage. They also demonstrated that SIX1 promotes cell cycle progression and proliferation by upregulation of cyclin D1. However, the role of SIX1 protein in prognostic evaluation and its relationship with survival in pancreatic cancer is unknown. The critical role of SIX1 in the initiation and progression of numerous cancers impelled us to study the clinical significance of SIX1 protein overexpression in pancreatic cancer.

In this study, we found evidence of SIX1 protein expression in PDAC patients and demonstrated its clinicopathological significance through prognostic evaluation of SIX1 overexpression in PDAC. The results revealed that SIX1 protein is frequently upregulated in PDAC compared with normal pancreatic tissue. SIX1 may be a good independent predictor of prognosis for patients with PDAC.

Section snippets

Clinical samples

A total of 148 tissue samples (103 PDAC and 45 normal pancreatic tissues) were collected from Shanghai Outdo Biotech Co. Ltd. and Tumor Tissue Bank, Yanbian University Medical College. All samples were routinely fixed in 10% buffered formalin and embedded in paraffin blocks. The study protocol was approved by the institutional review board of Yanbian University Medical College.

The pathological parameters, including age, gender, tumor location, tumor size, clinical stage, grading and

SIX1 protein expression in PDAC and normal pancreas

The immunofluorescence staining revealed that SIX1 protein showed strong positive signals in the cytoplasm and nucleoli of Panc-1 pancreatic cancer cells (Fig. 1), and the immunohistochemical staining showed that SIX1 protein was mainly positive in cytoplasm/perinucleus in PDAC, only small number of cells is nuclear staining pattern. The positive rate of SIX1 protein was 80.6% (83/103) in PDAC; being significantly higher than normal pancreatic tissue (24.4%, 11/45) (P < 0.01). Similarly, the

Discussion

The homeoprotein transcription factor SIX1 is located at chromosome 14q23, and it is a critical regulator of embryonic development that requires interaction with the EYA family of proteins to activate the transcription of genes involved in neurogenesis, myogenesis, and nephrogenesis (Laclef et al., 2003, Ozaki et al., 2004, Qamar et al., 2012, Relaix and Buckingham, 1999, Xu et al., 2003, Zheng et al., 2003). SIX proteins interact with EYA proteins for cooperative activation of their target

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Author contributions

AJ, YX, and SL participated in the study conception, design, case selection and immunohistochemical staining. TJ, ZL and AJ carried out data collection. YX, SL and HJ scored immunohistochemical staining. AJ, LL and ZL performed data analysis and wrote the manuscript. All the authors read and approved the final manuscript.

Acknowledgments

This study was supported by grants from the National Natural Science Funds of China (61371067 and 81160059), and The Projects of Research & Innovation of Jilin Youth Leader and Team (20130521017JH).

We thank Dr. Shuangping Liu (Department of Pathology, Yanbian University Medical College) for the help with the immunohistochemical scoring and statistical analysis.

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