USO1 expression is dysregulated in non-small cell lung cancer

Abstract

Background: USO1 vesicle transport factor (USO1) is a vesicular transport factor crucial for endoplasmic reticulum (ER) to Golgi transport and is required for transcytotic fusion and subsequent binding of the vesicles to the target membrane. USO1 has been studied in multiple cancers revealing high levels of expression and exerting its oncogenic role by increasing cell proliferation and evasion of apoptosis. Furthermore, multiple studies have implicated dysregulation of the Erk signalling pathway in the involvement of USO1 in multiple cancers. Overall survival (OS) in non-small cell lung cancer (NSCLC) remains low despite recent advances in treatments which are mainly due to the late stage of diagnosis and a significant cohort of patients lacking an available targeted therapy. The aim of this study was to investigate USO1 expression in NSCLC.

Methods: An in-house NSCLC tissue microarray (TMA) comprising (n=204 patients) was stained for USO1. Scoring intensity (H score) was used to interrogate for correlations between USO1 expression and established prognostic factors, and OS. Further evaluation of the expression of USO1 in NSCLC was done using multiple online datasets including Lung Cancer Explorer (LCE), UALCAN, GEPIA, KM plotter, TIMER2 and MuTarget.

Results: USO1, when highly expressed in lung adenocarcinomas (LUADs) leads to a significantly increased OS (P=0.028). There was no significant correlation between age, smoking status, lymph node status, tumour subgroup and stage. USO1 was significantly higher in patients with tumour size <5 cm compared to those ≥5 cm (P=0.016). Overexpression in LUAD occurred at an early stage being significantly upregulated in Stage 1 and N0 tumours. USO1's first neighbours, also involved in ER-Golgi transport have altered expression in LUAD and significantly impact overall survival. Overexpression occurred independently of commonly mutated genes in NSCLC and had no correlation with changes in the TME.

Conclusions: This study highlights the importance of USO1 and ER-Golgi vesicular transport system in LUAD. USO1 overexpression occurs as an early event in LUAD and independently of commonly mutated genes in NSCLC and therefore may represent an attractive diagnostic biomarker as well as a potential target for treatment.

Keywords: USO1; non-small cell lung cancer (NSCLC); overall survival (OS).

Figure 1

Representative examples of USO1 protein expression in NSCLC. (A) High level of expression (H score 300), (B) low level of expression (H score 100) and (C) negative staining (H score 0) in LUAD. (D) High level of expression (H score 300) & (E) low level of expression (H score 100), and (F) negative staining in LUSC. Strong positive staining in background inflammatory cells including lymphocytes and plasma cells can be seen in (C) & (F); magnification ×10; detection of USO1 protein by immunohistochemistry using monoclonal anti-p115 antibody; counterstain with haematoxylin II and Bluing Reagent. LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma.

Figure 2

Representative example of USO1 protein expression in normal lung parenchyma from (A), our TMA (IHC using monoclonal anti-p115 antibody) showing weak expression of USO1 and (B), from The Human Protein Atlas database (antibody CAB010108) showing no detected expression of USO1. Magnification ×10. TMA, tissue microarray; IHC, immunohistochemistry.

Figure 3

Kaplan-Meier survival analysis according to USO1 protein expression in (A) all histologies (n=204), (B) LUSC (n=108), (C) LUAD (n=82) (log-rank test). Probability of survival of (A) NSCLC, (B) LUSC, (C) LUAD: H-scores ≥188 (red line) and <188 (blue line). (A,B) there is no significant difference in survival when comparing all subgroups and the LUSC group. (C) In the LUAD group those with high levels of USO1 protein expression had significantly longer overall survival compared to those with low expression (P=0.0283). LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; NSCLC, non-small cell lung cancer.

Figure 4

USO1 mRNA and protein is overexpressed in NSCLC. Analysis of the TCGA dataset in UALCAN demonstrates that expression of USO1 is significantly increased in (A) LUAD (P=1.625×10⁻¹²) and (B) LUSC (P=1.545×10⁻³) compared to normal lung tissue. Reanalysis of the CPTAC (proteomic) dataset in UALCAN demonstrates that (C) USO1 total protein is significantly elevated in LUAD (P=2.207×10⁻⁴³) and (D) increased phosphorylation occurs at position S953 in these samples (P=3.867×10⁻²³). NSCLC, non-small cell lung cancer; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma.

Figure 5

High USO1 expression is associated with better OS and FP in LUAD. Kaplan-Meier curves. Survival analysis of USO1 expression in NSCLC in (A) all histologies, (B) LUAD, and (C) LUSC. FP analysis of USO1 expression in NSCLC in (D) all histologies, (E) LUAD, and (F) LUSC. All the above Kaplan-Meier survival curves were generated using Kaplan-Meier plotter web tool. FP, first progression; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma.

Figure 6

Analysis of CNV and mutations on USO1 expression in LUAD and LUSC. The effects of CNV on USO1 expression were analysed using cBioPortal for (A) LUAD and (B) LUSC. A screen for mutated genes which are correlated with altered USO1 expression was conducted using muTarget and validated using TIMER2. Significant mutated genes identified were (C) KRT47, (D) INSR and (E) OR10X1. CNV, copy number variations; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma.