Roles of the circular RNA circ-Foxo3 in breast cancer progression

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Circular RNAs (circRNAs) are an enigmatic class of RNA in biologic systems. CircRNAs are generated from genomic transcripts by forming covalently-closed loops, thereby joining the 5′ and 3′ ends. With alternative splicing the same primary RNA transcript yields diverse circRNAs. Specifically, a splice donor site is joined to a splice acceptor site further upstream of the primary transcript, resulting in the production of a circular RNA. In human cells, circRNAs are usually composed of 1–5 exons, and the exon 2 is often the upstream “acceptor” exon. These circRNAs generated from exons and are called exonic circRNAs. However exonic circRNAs have not been shown to code for proteins thus being categorized as non-coding RNAs. There are also circRNAs that are generated from introns and thus named intronic circRNAs. Circularization of transcripts was previously believed to be the result of erroneous splicing processes within cells. This idea was challenged by the observation that circRNAs were detected in various cell types in an evolutionarily conserved manner. All circRNAs are predominantly found in the cytoplasm. The copy number of circRNAs can be up to 10 times greater than that of associated linear RNAs, suggesting that these circRNAs possess potential biologic functions rather than accidental errors during splicing. However, the function of circRNAs remains unclear until recently. Studies have shown that some circRNAs harbor multiple binding sites for microRNAs, “sponging” microRNAs thereby serving as competitive inhibitors for microRNA functions. For example, the circRNA circ-ITCH contains binding sites for miR-7, miR-17, and miR-214 and represses the activity of these microRNAs.¹ To serve as a sponge, however, a circular RNA has to contain many microRNA binding sites and be expressed at sufficiently high levels in cytoplasm. The majority of circRNAs may not be in this category and their functions remain uncertain.

Most recently the function of a circRNA named circ-Foxo3 was well studied by a research group in Toronto led by Burton Yang. Specifically, this group found that circ-Foxo3 was downregulated in patient tumor samples and in a group of cancer cells, whereas it was upregulated in the cancer cells when the cells were induced to undergo apoptosis.² The authors showed that silencing endogenous circ-Foxo3 produced an opposite effect, while ectopic circ-Foxo3 induced cell apoptosis and inhibit progression of tumor xenografts. Interestingly, ectopic circ-Foxo3 played roles in increasing Foxo3 protein levels. The authors also demonstrated that circ-Foxo3 facilitated p53 ubiquitination and degradation by binding to both p53 and MDM2. As a consequence, this action decreased the capacity of MDM2 to induce Foxo3 ubiquitination and degradation, leading to increased Foxo3 protein. In addition, the authors identified the binding sites by computational algorithm and generated mutations in the binding sites of circ-Foxo3, which resulted in abolishment of the binding activity. They also used blocking oligos to prevent the binding activity. These approaches appeared to be valuable for studying circRNA functions, as evidenced that obstructing binding activity abolished circ-Foxo3 function.

Higher circRNA numbers were detected in normal breast mammary tissues than the tumor tissues. These numbers appear to be inversely correlated with the risk-of-relapse proliferation score for proliferating genes in breast cancer. Consistent with this notion, results from the Yang group suggest a role of circ-Foxo3 in tumor genesis and progression. In this regard, the involvement of circRNAs in breast cancer progression has been reported by others.³ Moreover, new results demonstrated important roles of Foxo3 in breast cancer progression. For example, downregulation of Foxo3 expression due to increased Akt activity or loss of PTEN is often observed in cancer development and thus Foxo3 has been classified as a tumor suppressor gene.⁴ In contrast, overexpression of Foxo3 decreases motility, invasiveness, and anchorage-independent growth.⁵ Given that post-transcriptional repression of Foxo3 expression occurs through microRNA targeting as well as other transcription mechanisms, the Yang group made efforts in figuring out mechanisms by which circ-Foxo3 regulates Foxo3 expression. The circRNA, pseudogene, and linear mRNA shared common sites for binding to 8 microRNAs. Interestingly, the Yang group showed that circ-Foxo3 could promote Foxo3 protein levels by binding to several microRNAs shared with the Foxo3 linear mRNA.⁶ In addition, the ectopic Foxo3 pseudogene and circ-Foxo3 played roles in suppressing cancer cell proliferation, survival and progression of breast cancer cells. Together, these results suggest that circ-Foxo3 can up-regulate Foxo3 protein levels by 2 different mechanisms. Importantly, results from Yang's group suggest that circ-Foxo3 and its relevant protein Foxo3 may be important for biologic functions. Indeed, using a mouse model of transverse aortic constriction (TAC) this group has demonstrated that levels of circ-Foxo3 in the cardiomyocytes regulate the overload-induced cardiomyopathy and hence heart functions.⁷

In summary, studies from the Yang group showed that circ-Foxo3 regulates cellular functions of tissue cells and suppresses cancer cell progression. In addition, novel approaches created by this group are valuable for studying circRNA functions.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.