Deregulated expression of hnRNP A/B proteins in human non-small cell lung cancer: parallel assessment of protein and mRNA levels in paired tumour/non-tumour tissues

<p>Abstract</p> <p>Background</p> <p>Heterogeneous nuclear ribonucleoproteins (hnRNPs) of the A/B type (hnRNP A1, A2/B1, A3) are highly related multifunctional proteins participating in alternative splicing by antagonising other splicing factors, notably ASF/SF2. The altered expression pattern of hnRNP A2/B1 and/or splicing variant B1 alone in human lung cancer and their potential to serve as molecular markers for early diagnosis remain issues of intense investigation. The main objective of the present study was to use paired tumour/non-tumour biopsies from patients with non-small cell lung cancer (NSCLC) to investigate the expression profiles of hnRNP A1, A2/B1 and A3 in conjunction with ASF/SF2.</p> <p>Methods</p> <p>We combined western blotting of tissue homogenates with immunohistochemical examination of fixed tissue sections and quantification of mRNA expression levels in tumour versus adjacent normal-looking areas of the lung in the same patient.</p> <p>Results</p> <p>Our study, in addition to clear evidence of mostly uncoupled deregulation of hnRNPs A/B, has revealed hnRNP A1 to be the most deregulated protein with a high frequency of over-expression (76%), followed by A3 (52%) and A2/B1 (43%). Moreover, direct comparison of protein/mRNA levels showed a lack of correlation in the case of hnRNP A1 (as well as of ASF/SF2), but not of A2/B1, suggesting that different mechanisms underlie their deregulation.</p> <p>Conclusion</p> <p>Our results provide strong evidence for the up-regulation of hnRNP A/B in NSCLC, and they support the existence of distinct mechanisms responsible for their deregulated expression.</p

Deciphering the role of proteins with ability to bind hnRNA and/or mRNA and their deregulated expression in lung cancer

The main goal of the present Thesis was to determine the molecular characteristics of the deregulated expression of RNA binding proteins (RBPs), in the human lung cancer, in paired biopsies taken from cancerous and normal adjacent tissue from the same patient. The study focused in members of hnRNP proteins family belonging to the hnRNP A/B subtype (A1, A2/B1 and A3), along with other hnRNPs (hnRNP K/J) as well as the splicing factor ASF/SF2 with a known antagonistic role to hnRNPs in the alternative splicing. In total, 21 biopsy pairs were examined from patients with non-small cell lung cancer (NSCLC) (in collaboration with Dr.Ch. Valavanis, “Metaxa” hospital, in the frame of the PENED program). Western blotting and quantitative Real-Time PCR were applied for the semi-quantitative estimation of protein levels in combination with the quantitive estimation of mRNA levels, respectively. This approach allowed the direct comparison of protein and mRNA patterns of expression in cancer/normal tissue from the same patient. To summarize the results, the comparison of cancer related alterations of hnRNP proteins and ASF/SF2 pointed to the lack of correlation between protein and mRNA levels, with the exception of the hnRNP A2/B1. Moreover, the level of overexpression detected for hnRNP A1 and ASF/SF2 in the cancerous tissue, did not correlate with changes in the mRNA level. The results support the existence of distinct molecular mechanisms regulating the expression of closely related hnRNP A/B subtype proteins. In the course of this project new tools, useful in study of hnRNPs, were produced, refering to the expression of recombinant hnRNP A2 and A3 proteins. The application of these new tools, in a series of pull-down experiments performed in lung cell lines extracts, led to the identification of new interactions among hnRNP proteins, in hnRNP and mRNP complexes. The use of these tools also allowed us to investigate the possibility of establishing the hnRNP proteins as biomarker for the early detection of lung cancer. Experiments applying the recombinant proteins hnRNP A/B, as specific antigens, gave clear evidence for the presence of autoantibodies targeting those proteins, in the blood serum of patients with cancer, a finding that rises the perspective of using these proteins, in combination with other biomarkers, as a reliable marker for malignant detection in early stages of lung cancer