Abstract
BackgroundMetastasis causes the majority of cancer-related deaths worldwide. Increasing studies have revealed that circRNAs are associated with the carcinogenesis and metastasis of many cancers. Nevertheless, the biological mechanisms of circRNAs in breast cancer (BC) liver metastasis remain extremely ambiguous.MethodsIn this study, we identified circROBO1 from three pairs of primary BC and metastatic liver sites by RNA sequencing. FISH assays and RT-qPCR were conducted to validate the existence and expression of circROBO1. The oncogenic role of circROBO1 was demonstrated both in vitro and in vivo. Western blot, ChIP, RIP, RNA pull-down, and dual-luciferase reporter assays were used to confirm the interaction of the feedback loop among circROBO1, miR-217-5p, KLF5, and FUS. Meanwhile, the regulation of selective autophagy was investigated by immunofluorescence, CoIP, and western blot.ResultsIn this study, upregulated expression of circROBO1 was found in BC-derived liver metastases and was correlated with poor prognosis. Knockdown of circROBO1 strikingly inhibited the proliferation, migration, and invasion of BC cells, whereas overexpression of circROBO1 showed the opposite effects. Moreover, overexpression of circROBO1 promoted tumor growth and liver metastasis in vivo. Further research revealed that circROBO1 could upregulate KLF5 by sponging miR-217-5p, allowing KLF5 to activate the transcription of FUS, which would promote the back splicing of circROBO1. Therefore, a positive feedback loop comprising circROBO1/KLF5/FUS was formed. More importantly, we found that circROBO1 inhibited selective autophagy of afadin by upregulating KLF5.ConclusionsOur results demonstrated that circROBO1 facilitates the carcinogenesis and liver metastasis of BC through the circROBO1/KLF5/FUS feedback loop, which inhibits the selective autophagy of afadin by suppressing the transcription of BECN1. Therefore, circROBO1 could be used not only as a potential prognostic marker but also as a therapeutic target in BC.
Highlights
Metastasis causes the majority of cancer-related deaths worldwide
CircROBO1 correlates with breast cancer (BC) liver metastasis To identify the differentially expressed circRNAs in liver metastases derived from BC, we performed highthroughput RNA sequencing (RNA-seq) in 3 pairs of BC primary tumors and matched liver metastases
Among the upregulated circRNAs, circROBO1, which consists of four exons, was our main focus, and its back spliced junction site of exon 8 and exon 5 was verified by Sanger sequencing in BT-549 cells (Fig. 1c)
Summary
Metastasis causes the majority of cancer-related deaths worldwide. The biological mechanisms of circRNAs in breast cancer (BC) liver metastasis remain extremely ambiguous. According to a recent report, the survival rate at 5 years for primary BC is almost 99%. Approximately one-third of patients with BC present distant nonnodal metastasis, Wang et al Molecular Cancer (2022) 21:29 which decreases their 5-year survival rate to 23% [1]. If liver metastasis is left untreated, the patients’ survival time ranges from approximately 4 to 8 months [3]. Current treatments for BC-derived liver metastasis are primarily based on systemic hormones and/or chemotherapy [4, 5], which only might extend the survival of patients to approximately 18-24 months [6]. Little is known about the concrete mechanism by which the invasive capability of cancer cells is enhanced in BC progression, which might be a promising target for precise antimetastatic treatment
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