Heterogeneous nuclear ribonucleoprotein K is over expressed, aberrantly localised and is associated with poor prognosis in colorectal cancer

Abstract

Heterogeneous ribonucleoprotein K (hnRNP K) is a member of the hnRNP family which has several different cellular roles including transcription, mRNA shuttling, RNA editing and translation. Several reports implicate hnRNP K having a role in tumorigenesis, for instance hnRNP K increases transcription of the oncogene c-myc and hnRNP K expression is regulated by the p53/MDM 2 pathway. In this study comparing normal colon to colorectal cancer by proteomics, hnRNP K was identified as being overexpressed in this type of cancer. Immunohistochemistry with a monoclonal antibody to hnRNP K (which we developed) on colorectal cancer tissue microarray, confirmed that hnRNP K was overexpressed in colorectal cancer (P<0.001) and also showed that hnRNP K had an aberrant subcellular localisation in cancer cells. In normal colon hnRNP K was exclusively nuclear whereas in colorectal cancer the protein localised both in the cytoplasm and the nucleus. There were significant increases in both nuclear (P=0.007) and cytoplasmic (P=0.001) expression of hnRNP K in Dukes C tumours compared with early stage tumours. In Dukes C patient's good survival was associated with increased hnRNP K nuclear expression (P=0.0093). To elaborate on the recent observation that hnRNP K is regulated by p53, the expression profiles of these two proteins were also analysed. There was no correlation between hnRNP K and p53 expression, however, patients who presented tumours that were positive for hnRNP K and p53 had a poorer survival outcome (P=0.045).

Figure 1

Coomassie-stained 2D SDS-PAGE gels showing whole-cell lysates of normal colon (A) and colorectal tumour samples (B). Circle highlights a protein spot present in the tumour samples but not in the normal tissue, which was identified as hnRNP K by MALDI-TOF MS analysis. Molecular weight (Mw) and pI values are shown.

Figure 2

Immunoblot showing specificity of the hnRNP K monoclonal antibody. Whole-cell lysates of CAL U1 and HT 29 reveal the monoclonal antibody recognises a single band, migrating at the expected molecular weight for hnRNP K by Western blotting.

Figure 3

Immunoreactivity for hnRNP K in normal colon and colorectal cancer. (A) In normal colon, hnRNP K immunoreactivity is exclusively present in the nuclei of crypt epithelial cells. (B) Strong nuclear and cytoplasmic staining in primary colorectal cancer. (C) Strong nuclear and weak cytoplasmic staining in primary colorectal neoplasm.

Figure 4

Mean score of hnRNP K in normal colon, primary tumour and metastatic tissues. (A) Total mean scores showed that there was a significant increase in hnRNP K expression when comparing normal colon to primary colorectal cancer (P<0.001). Subsequently mean scores were evaluated based on cellular localisation, nuclear (B) and cytoplasm (C). (B) As tumour stage progressed hnRNP K nuclear expression significantly decreased (normal to tumour P<0.001 and tumour to metastasis P=0.006). (C) No hnRNP K expression was observed in the cytoplasm of normal tissue but there was a significant increase in cytoplasmic immunoreactivity (P<0.001) in primary tumours. No difference was observed in mean hnRNP K cytoplasmic score for the tumour to metastasis transition.

Figure 5

The mean nuclear and cytoplasmic score in the different stages of colorectal cancer. There are significant increases in both nuclear (A) and cytoplasmic (B) hnRNP K in Dukes C tumours (P=0.007 and 0.001, respectively).

Figure 6

Comparison of survival in patients whose tumours with a high nuclear hnRNP K expression and those tumours with a low or moderate nuclear hnRNP K expression. There is poorer survival in those patients whose tumours with a low or moderate hnRNP K nuclear expression compared with those patients whose tumours have a high nuclear expression (log rank=6.77, P=0.0093).

Figure 7

Survival analysis patients who presented tumours that were hnRNP K+/p53+had a poor survival outcome compared to patients with tumours that were either negative for both proteins or, p53+/hnRNP K− or p53−/hnRNP K+(log rank=4.001, P=0.045).