Elsevier

Human Pathology

Volume 42, Issue 3, March 2011, Pages 393-402
Human Pathology

Original contribution
The expression profile of RNA-binding proteins in primary and metastatic colorectal cancer: relationship of heterogeneous nuclear ribonucleoproteins with prognosis

https://doi.org/10.1016/j.humpath.2010.08.006Get rights and content

Summary

The heterogeneous nuclear ribonucleoproteins are a group of RNA-binding proteins with a range of key cellular functions, which are dysregulated in tumorigenesis including regulation of translational and RNA processing. The aims of this study were to define the heterogeneous nuclear ribonucleoprotein expression profile in primary and metastatic colorectal cancer and to establish the clinicopathologic significance of this expression. A tissue microarray containing 515 primary colorectal cancers, 224 lymph node metastasis of colorectal cancer, and 50 normal colon samples was immunostained for 6 heterogeneous nuclear ribonucleoproteins. Heterogeneous nuclear ribonucleoprotein I, heterogeneous nuclear ribonucleoprotein K, and heterogeneous nuclear ribonucleoprotein L displayed the most frequent strong immunoreactivity in primary colorectal tumor samples. Heterogeneous nuclear ribonucleoprotein A1 (P < .001) and heterogeneous nuclear ribonucleoprotein U (P = .003) showed significant alterations in nuclear expression in tumors compared with normal colonic epithelium, whereas heterogeneous nuclear ribonucleoprotein A1 (P = .001), heterogeneous nuclear ribonucleoprotein I (P < .001), and heterogeneous nuclear ribonucleoprotein K (P < .001) all showed significant aberrant cytoplasmic immunoreactivity in tumor cells. There were also significant differences in cytoplasmic immunoreactivity between the primary tumor and the corresponding lymph node metastasis for heterogeneous nuclear ribonucleoprotein A1 (P = .001), heterogeneous nuclear ribonucleoprotein I (P < .001), and heterogeneous nuclear ribonucleoprotein K (P = .001). Nuclear heterogeneous nuclear ribonucleoprotein H (χ2 = 72.1; P < .001), cytoplasmic heterogeneous nuclear ribonucleoprotein I (χ2 = 28.1; P < .001), and cytoplasmic heterogeneous nuclear ribonucleoprotein K (χ2 = 13.2; P = .04) all showed significant associations with tumor stage. There was a significant relationship between strong nuclear heterogeneous nuclear ribonucleoprotein H expression and survival (χ2 = 14.97; P < .001). This study has defined the expression profile of heterogeneous nuclear ribonucleoproteins in colorectal cancer and shown that there are significant alterations in both expression and subcellular localization of individual heterogeneous nuclear ribonucleoproteins in this type of tumor.

Introduction

The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a group of RNA-binding proteins whose main function is to bind newly translated messenger RNA (mRNA), in particular pre-mRNA, via RNA recognition motifs and regulate mRNA stability [1], [2], [3]. As a consequence, the hnRNPs have an important involvement in a diverse range of key biologic processes including RNA processing, DNA repair, transcription factor activity, translational control, regulation of alternative splicing, regulation of apoptosis, and control of cell proliferation [4], [5], [6], [7], [8], [9], [10]. Most hnRNPs are normally localized to the nucleus; however, some may shuttle between the nucleus and cytoplasm because several hnRNPs contain a nuclear export signal [11].

Despite the involvement of individual hnRNPs in many of the key biologic processes that can be disrupted in tumorigenesis, there have been relatively few studies of hnRNPs in cancer [12]. hnRNPK is the most widely studied, and its expression and the significance of its expression have been investigated in several types of cancer including colorectal cancer, prostate cancer, pancreatic cancer, oral cancer, nasopharyngeal cancer, and cervical cancer [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23].

In colorectal cancer, aberrant cytoplasmic localization of hnRNPK has been observed [13], and an alternatively spliced hnRNPK mRNA transcript has been identified in colorectal cancer [14]. The expression of hnRNPK has also been associated with poor outcome in prostate cancer [15]. hnRNPK has also been proposed as a regulator of androgen receptor translation [16] and a mediator of docetaxel therapy in androgen-resistant prostate cancer and has also been suggested as a target for therapy in prostate cancer [17]. In pancreatic cancer, increased cytoplasmic expression of hnRNPK has been observed and that hnRNPK was associated with mutant p53 [18]. The presence of hnRNPK has been identified as a marker of oral leuokoplakia, and its expression has also been associated with poor prognosis in established oral squamous carcinoma [19]. hnRNPK is also involved with the regulation of telomerase reverse transcriptase in oral squamous carcinoma cells [20]. In nasopharyngeal carcinoma, hnRNPK has been associated with poor prognosis and also shown to function to increase thymidine phophorylase mRNA stability, suggesting that hnRNPK may be a therapeutic target in this type of cancer [21], [22]. hnRNPH has recently been found to have increased nuclear expression in head and neck squamous carcinoma and also to interact with the raf transcription pathway, thus inhibiting apoptosis [24]. hnRNPA2B1 has been identified in lung cancer and proposed as a putative biomarker [25], [26], whereas hnRNPA1 has been shown to be a potential biomarker of colorectal cancer based on differential proteomic studies [27].

In this comprehensive study, the expression and cellular localization of 6 hnRNPs, namely, hnRNPA1, hnRNPH, hnRNPI, hnRNPL, hnRNPK, and hnRNPU, have been evaluated in a large series of primary colorectal cancers, corresponding lymph node metastasis, and normal colonic epithelium, and the clinicopathologic significance of expression of these proteins, including their relationship to survival, has been established.

Section snippets

Antibodies

Primary hnRNP antibodies (Table 1) to hnRNPA1, hnRNPI, hnRNPL, and hnRNPU were obtained from Abcam (Cambridge, UK), and an antibody to hnRNPH was bought from Sigma-Aldrich (Gillingham, Dorset, UK). The monoclonal antibody to hnRNPK was produced in Murray's laboratory, and its development and characterization, including its use in immunohistochemistry, have previously been described [13].

Design and construction of colorectal cancer tissue microarray

All cases were selected from the Aberdeen Colorectal Tumor Bank. In total, tumor samples from 515 patients

hnRNP expression in normal colon

In normal colonic epithelium, there was consistent strong nuclear expression for each of the hnRNPs except hnRNPH, which showed weak-to-moderate intensity immunoreactivity (Fig. 1, Fig. 2). There was no apparent gradient of hnRNP immunoreactivity from crypt base to surface epithelium. Cytoplasmic immunoreactivity was not observed in normal colonic epithelium except for one case, which showed hnRNPU immunoreactivity.

hnRNP expression in primary and metastatic colorectal cancer

All the hnRNPs studied showed nuclear localization, with hnRNPs I, K, and L

Discussion

The hnRNPs are a group of RNA-binding proteins that participate in a wide range of key cellular functions involving many of the pathways implicated, disrupted, or dysregulated in tumor development and progression [1], [2], [3]. Most of the functions that have been identified for the hnRNPs, including RNA processing and regulation of translation, DNA repair in particular response to ionizing radiation, control of alternative splicing, and response to hypoxia, are relevant in the context of

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    This research was funded by the Association for International Cancer Research, St Andrews, UK and the University of Aberdeen Development Trust, Aberdeen, UK.

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