The Expression of the Ubiquitin Ligase SIAH2 (Seven <i>In Absentia</i> Homolog 2) Is Increased in Human Lung Cancer

<div><p>Objectives</p><p>Lung cancer is the leading cause of cancer-related deaths worldwide. Overall 5-year survival has shown little improvement over the last decades. Seven in absentia homolog (SIAH) proteins are E3 ubiquitin ligases that mediate proteasomal protein degradation by poly-ubiquitination. Even though SIAH proteins play a key role in several biological processes, their role in human cancer remains controversial. The aim of the study was to document SIAH2 expression pattern at different levels (mRNA, protein level and immunohistochemistry) in human non-small cell lung cancer (NSCLC) samples compared to surrounding healthy tissue from the same patient, and to analyse the association with clinicopathological features.</p><p>Materials and Methods</p><p>One hundred and fifty-two samples from a patient cohort treated surgically for primary lung cancer were obtained for the study. Genic and protein expression levels of SIAH2 were analysed and compared with clinic-pathologic variables.</p><p>Results</p><p>The present study is the first to analyze the SIAH2 expression pattern at different levels (RNA, protein expression and immunohistochemistry) in non-small cell lung cancer (NSCLC). We found that SIAH2 protein expression is significantly enhanced in human lung adenocarcinoma (ADC) and squamous cell lung cancer (SCC). Paradoxically, non-significant changes at RNA level were found, suggesting a post-traductional regulatory mechanism. More importantly, an increased correlation between SIAH2 expression and tumor grade was detected, suggesting that this protein could be used as a prognostic biomarker to predict lung cancer progression. Likewise, SIAH2 protein expression showed a strong positive correlation with fluorodeoxyglucose (2-deoxy-2(¹⁸F)fluoro-D-glucose) uptake in primary NSCLC, which may assist clinicians in stratifying patients at increased overall risk of poor survival. Additionally, we described an inverse correlation between the expression of SIAH2 and the levels of one of its substrates, the serine/threonine kinase DYRK2.</p><p>Conclusions</p><p>Our results provide insight into the potential use of SIAH2 as a novel target for lung cancer treatment.</p></div

SIAH2 mRNA expression levels in NSCLC.

<p>Total RNA was extracted from tissues, integrity evaluated (all the samples included showed a RIN above 9) and changes in expression of SIAH2 in tumor samples compared to normal lung samples from the same patient analyzed by qPCR and expressed as a fold-change. Amplification efficiencies were validated and normalized against β-actin and HPRT, and fold change in gene expression was calculated using the 2^−ΔΔCt method. Results represent the mean ± SD.</p

Scatter plot showing results of Pearson´s correlation analysis.

<p>(A) Positive correlation between SIAH2 protein expression and 18FDG uptake (measured as maximum standardized uptake value-SUVmax) in primary NSCLC (<i>p</i> = 0.014), r2 = 0.13). (B) Positive correlation between increasing tumor size and 18FDG uptake on PET/CT scans (<i>p</i> = 0.012, r2 = 0.12).</p

Patient characteristics.

<p>Patient characteristics.</p

SIAH2 protein expression correlates with expression of DYRK2.

<p>(A) Representative images of adenocarcinoma or squamous cell carcinoma, and adjacent normal tissue stained with DYRK2 antibody (x100). (B) BEAS-2B cells were cultured with or without serum during 7 days, lysed and protein expression was evaluated by immunoblot with the indicated antibodies (upper panel) and mRNA expression by qPCR (lower panel). Representative blot out of three independent experiments and the positions and molecular weights (in kDa) are indicated.</p

SIAH2 protein expression in human lung cancer compared to surrounding healthy tissue.

<p>(A, B) Total proteins were extracted from ADC or SDC specimens and the corresponding normal lung tissue, SIAH2 protein expression analyzed by western blot and the bands quantified by densitometry after normalization to actin signal intensities. The results are expressed as relative OD expression and represent the mean ± SD. ** <i>p</i> < 0.001. (C) Fold induction representation of SIAH2 in tumor samples compared to normal lung samples from the same patient. Results represent the mean ± SD. (D) Results from 16 patients (8 ADC and 8 SCC) selected to represent a range of differential mRNA expression between normal lung tissue (N) and tumor (T). SIAH2 protein expression was analyzed by immunoblots (upper panel) and mRNA expression by qPCR (lower panel). Data are mean ± SD of n = 3 experiments. ** <i>p</i> < 0.001 and *** <i>p</i> < 0.0001.</p