Comparative O-GlcNAc Proteomic Analysis Reveals a Role of O-GlcNAcylated SAM68 in Lung Cancer Aggressiveness.

Chia-Hung Lin,Teh-Ying Chou,Shu-Ying Wang,Chen-Chung Liao,Yi-Chen Yeh,Mei-Yu Chen,Chia-Yi Peng

doi:10.3390/cancers14010243

Chia-Hung Lin, Teh-Ying Chou + Show 5 more

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https://doi.org/10.3390/cancers14010243

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Abstract

Simple SummaryLung cancer claims the most lives annually among cancers; to date, invasion and metastasis still pose challenges to effective treatment. O-GlcNAcylation, an enzymatic modification of proteins after biosynthesis, modulates the functions of many proteins. Aberrant O-GlcNAcylation is linked to pathogenic mechanisms of cancer, including invasion and metastasis. However, little is known about the profile of O-GlcNAcylated proteins involved in cancer aggressiveness. Here, by comparing profiles of O-GlcNAcylated proteins from two lung cancer cell lines different in their invasive potential, we identified candidates for O-GlcNAcylated proteins that may be involved in cancer aggressiveness. One of these candidates, SAM68, was further characterized. Results confirmed O-GlcNAcylation of SAM68; functional analyses on SAM68 with mutations at O-GlcNAcylation sites suggested a role of O-GlcNAcylated SAM68 in modulating lung cancer cell migration/invasion. Future elucidation of the functional significance of differential O-GlcNAcylation of proteins identified in this study may provide new insights into mechanisms of lung cancer progression.O-GlcNAcylation is a reversible and dynamic post-translational protein modification catalyzed by O-GlcNAc transferase (OGT). Despite the reported association of O-GlcNAcylation with cancer metastasis, the O-GlcNAc proteome profile for cancer aggressiveness remains largely uncharacterized. Here, we report our comparative O-GlcNAc proteome profiling of two differentially invasive lung adenocarcinoma cell lines, which identified 158 down-regulated and 106 up-regulated candidates in highly invasive cells. Among these differential proteins, a nuclear RNA-binding protein, SAM68 (SRC associated in mitosis of 68 kDa), was further investigated. Results showed that SAM68 is O-GlcNAcylated and may interact with OGT in the nucleus. Eleven O-GlcNAcylation sites were identified, and data from mutant analysis suggested that multiple serine residues in the N-terminal region are important for O-GlcNAcylation and the function of SAM68 in modulating cancer cell migration and invasion. Analysis of clinical specimens found that high SAM68 expression was associated with late cancer stages, and patients with high-OGT/high-SAM68 expression in their tumors had poorer overall survival compared to those with low-OGT/low-SAM68 expression. Our study revealed an invasiveness-associated O-GlcNAc proteome profile and connected O-GlcNAcylated SAM68 to lung cancer aggressiveness.

Highlights

Lung cancer is by far the most common cause of cancer death worldwide; globally reported deaths from lung cancer were around 180,000 in 2020 [1]
Differential O-GlcNAcylated Proteins in Lung Adenocarcinoma Cell Lines with Low and Aiming at identifying O-GlcNAcylated proteins related to cancer invasiveness, we employed CL1-1 and CL1-5, which are two well-established human lung adenocarcinoma cell lines with differential invasiveness (Figure 1A)
We examined the amounts of WGAbound IQ Motif Containing GTPase Activating Protein 1 (IQGAP1), Lysine-specific histone demethylase 1 (LSD1), Metastasis Associated 1 Family, Member 2 (MTA2), Src-Associated in Mitosis 68 KDa Protein (SAM68), and Nucleophosmin (NPM) in nuclear fractions and the amounts of wheat germ agglutinin (WGA)-bound Rho-associated protein kinase 1 (ROCK-1) and α-Enolase in cytosolic fractions from CL1-1 and CL1-5 cells

Summary

Introduction

Lung cancer is by far the most common cause of cancer death worldwide; globally reported deaths from lung cancer were around 180,000 in 2020 [1]. Adenocarcinoma accounting for ~40% and ~70% of the male and female lung cancers in Taiwan, is the prevailing histological type of NSCLC and the most common primary lung malignancy in patients who have never smoked [2,3,4]. The prognosis of lung cancer remains poor, and the overall 5-year survival rate is only 10–20% [1]. The mortality mainly results from metastasis, which is a progression requiring cancer cells to undergo intravasation, survival in circulation, extravasation, and colonization [5]. Cancer invasion—a process involving disruption of the surrounding extracellular matrix and increasing cell motility—is the initial and a critical step for metastasis. A complete understanding of the molecular mechanisms underlying the regulation of cancer invasiveness remains elusive

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