Abstract

Over the last few decades, circular RNAs (circRNAs) have emerged as critical drivers of cancer progression, yet their precise and comprehensive role in oncogenesis remains to be fully elucidated. Owing to their structural stability, broad expression profile, and complex molecular interactions, circRNAs are capable of influencing tumor development across multiple cancer hallmarks, positioning them as key regulatory molecules. Among these, circITGB6 has recently garnered significant attention for its established role in TGFB1-induced epithelial-mesenchymal transition (EMT), a process strongly linked to enhanced metastatic potential. This review aims to discuss the integral role of circRNAs in oncogenesis, with a specific focus on circITGB6. We provide examples of recent advancements in research that accentuate the importance of circRNAs throughout the stages of tumor development: initiation, promotion, progression, and metastasis. Specifically, we detail the mechanism by which circITGB6 enhances EMT and promotes cancer cell migration and invasion in colorectal cancer by interacting with IGF2BP3 to increase podoplanin PDPN mRNA stability. This mechanism highlights its potential as a vital diagnostic marker and therapeutic target. Additionally, the general function of circRNAs in oncogenesis is discussed concerning their broader application as promising biomarkers and therapeutic strategies in personalized cancer care.

Content

RNA Splicing and the Discovery of circRNAs
Ribonucleic acid (RNA) splicing is a fundamental process in gene expression, meticulously orchestrated by the spliceosome ribonucleoprotein complex. While typically associated with the removal of introns and the ligation of exons from pre-mRNAs to generate mature messenger RNAs (mRNAs), various non-canonical splicing events also occur. Among these, backsplicing is the mechanism leading to the expression of circular RNAs (circRNAs) ¹. CircRNAs are distinct, covalently closed RNA molecules formed when a 3’ splice site joins a 5’ splice site, a configuration that confers protection from exonuclease activity. Due to their circular structure, circRNAs naturally lack both the 5’ cap and the 3’ poly(A) tail ². Historically, this structure led them to be dismissed as mere splicing artifacts.

Functional Significance in Cancer
Emerging evidence now shows that circRNAs play important roles in diverse physiological and pathological processes, including cancer initiation and progression ³. Their high stability enables circRNAs to accumulate in a cell type-specific manner—often at levels exceeding those of their linear isoforms—allowing them to act as functional regulators or disease biomarkers. In cancer, circRNAs participate in key cellular pathways such as proliferation, apoptosis, genomic instability, metastatic spread, and treatment resistance ⁴. This functional versatility stems from their ability to interact with multiple molecular partners, including microRNAs (miRNAs), RNA-binding proteins (RBPs), and even DNA ⁵. For example, circRNAs can sequester miRNAs and thereby relieve repression of their target mRNAs, or serve as scaffolds for assembling protein complexes involved in signal transduction ⁶. In addition, some circRNAs form RNA:DNA hybrids (circR-loops) that influence transcription and genome stability by altering chromatin accessibility or interfering with replication fork progression ⁷. Altered availability of proteins involved in circRNA biogenesis, such as splicing factors and RNA helicases, also correlates with circRNA expression changes and cancer development ⁸.

circRNAs in Oncogenesis: Mechanisms and Stages
Oncogenesis is broadly characterized by three main processes—initiation, promotion, and progression—each defined by distinct molecular and cellular events ⁹. A fourth stage, treatment resistance, is also relevant, as circRNAs represent potential therapeutic targets for overcoming acquired resistance mechanisms ¹⁰ (Figure 1).

Figure 1. Roles of circRNAs at different stages of oncogenesis. The functional roles of circular RNAs in various stages of oncogenesis are explained herein. Oncogenesis is divided into initiation, promotion, progression and metastasis. The presence of particular circular RNAs (circRNAs) is shown at each of these stages of oncogenesis. The inhibition of targets is depicted by block-ended arrows while normal arrows depict increase in expression or activity of targets. The circRNAs are coloured according to their interacting partners type by which they exert their effects. Key terms include EMT, which stands for epithelial-to-mesenchymal transition; PDPN, which stands for podoplanin; and VEGFA, which stands for vascular endothelial growth factor A ¹¹.

Initiation and Promotion
Initiation: Genomic Instability and Mutations
CircRNAs can contribute to the onset of oncogenesis by regulating gene expression and promoting genomic instability. A critical example is circMLL(9,10), derived from the gene KMT2A. This circRNA interacts with genomic DNA to form circR-loops, a mechanism that blocks the proteasome and disrupts RNA polymerase II elongation ¹². This disruption leads to increased genomic instability, resulting in cancer-predisposing mutations and chromosomal rearrangements ¹². This process, termed endogenous RNA-directed DNA damage (ER3D), exemplifies a circRNA's role in promoting oncogenic mutations ⁴ and is currently the only known example of an endogenous RNA directly inducing cancer development. Beyond circMLL(9,10), other nuclear circRNAs, such as circSMARCA5, are implicated in DNA repair and transcription, suggesting a general role for circRNAs in cancer initiation through mechanisms that disrupt genomic integrity and alter gene expression ¹³.

Promotion: Proliferation and Apoptosis Regulation
In the promotion stage, circRNAs regulate cell fate decisions like proliferation and apoptosis. circACTN4 functions as an oncogene by binding to FUBP1, this interaction prevents the inhibitory action of FARP2, thereby enhancing FUBP1-mediated transcriptional activation of MYC ¹⁴. The enhanced expression of MYC, a well-established driver of tumor growth ¹⁵, promotes cell proliferation and tumor progression, as observed in mouse models of breast cancer ¹⁴. Conversely, circFOXO3 generally plays a tumor-suppressive role by regulating apoptosis. It forms a complex with the pro-apoptotic transcription factor and, a protein that promotes the degradation of TP53 ¹⁶. While the circFOXO3-FOXO3-MDM2 complex ultimately leads to the ubiquitination and degradation of TP53 and suppresses apoptosis ¹⁷, circFOXO3 levels are typically low in tumor samples compared to normal tissues ¹⁸. Notably, its levels increase during stress-induced apoptosis, implying a role in regulating the cell death machinery ¹⁸.

Progression and Metastasis
Progression: Angiogenesis Regulation
Angiogenesis, the formation of new blood vessels, is crucial for sustaining tumor growth ¹⁹ and is a target for antitumor drugs. CircRNAs are key regulators of this process. circSMARCA5 exhibits a dual role in angiogenesis. It acts as a sponge for the serine-rich/arginine-rich splicing factor 1 SRSF1, shifting the splicing of pro-angiogenic isoforms of vascular endothelial growth factor A VEGFA to anti-angiogenic ones, thereby suppressing angiogenesis ²⁰. However, circSMARCA5 can also show protumoral activity. Its repression in lung adenocarcinoma leads to decreased levels of epidermal growth factor receptor EGFR, a mediator of angiogenesis in the tumor and its microenvironment ²¹.

Metastasis: Epithelial-Mesenchymal Transition (EMT)
Metastasis is a highly organized process involving EMT, intravasation, circulation, and distant colonization ²². EMT is a pivotal event in progression where epithelial cells lose polarity and gain the motility and invasive properties necessary for migration ²³. CircRNAs are closely involved in regulating EMT. circSKA3 is highly expressed in colorectal cancer and promotes EMT by interacting with the transcriptional factor. This interaction inhibits the ubiquitination and subsequent degradation of, keeping it stable and active to induce EMT and tumor growth ²⁴.

circITGB6 in CRC Progression
The TGF-β–circITGB6–PDPN axis is a key signaling pathway regulating EMT. TGFB1 signaling promotes the generation of circITGB6, which in turn enhances the stability of PDPN (podoplanin) mRNA and thereby potentiates the EMT process ²⁵. This coordinated molecular crosstalk highlights how this axis governs cellular plasticity, detailing both its regulatory mechanisms and resulting consequences.

Interaction between circITGB6 and IGF2BP3
circITGB6 promotes tumor metastasis by stabilizing PDPN mRNA through its interaction with IGF2BP3, an RNA-binding protein involved in RNA metabolism, mRNA translation, and intracellular localization. IGF2BP3 associates with metastasis-related transcripts, including PDPN, ultimately increasing their expression ²⁶. In this context, circITGB6 serves as a critical mediator facilitating the interaction between IGF2BP3 and PDPN mRNA, leading to increased mRNA stability and elevated protein levels. This mechanism is independent of transcriptional regulation, as circITGB6 and IGF2BP3 do not affect PDPN pre-mRNA levels. Instead, circITGB6 increases the binding affinity of IGF2BP3 for PDPN mRNA, thereby enhancing its stability and sustaining its expression ²⁵ (Figure 2). Upregulation of PDPN, a protein central to cell migration and invasion, is a key event in EMT, which is characterized by the loss of epithelial markers such as E-cadherin and the gain of mesenchymal markers such as N-cadherin, enabling cancer cells to adopt migratory and invasive phenotypes ²⁷. The TGF-β/circITGB6/PDPN pathway induces EMT through increased PDPN expression; circITGB6 and PDPN, together with heightened TGFB1 signaling, show higher expression in metastatic sites compared with matched primary tumors. Beyond enhancing PDPN expression, this pathway contributes to reduced E-cadherin and increased N-cadherin levels, further strengthening cancer cell metastatic capacity ²⁸. Moreover, circITGB6-driven metastasis has also been demonstrated in lung cancer brain metastases, where both circITGB6 and PDPN exhibit higher expression than in matched primary tumors. This overexpression correlates with increased PDPN mRNA stability and enhanced cell migration, reinforcing the central role of the circITGB6/PDPN pathway in brain metastasis ²⁵.

Figure 2. Schematic illustration of circITGB6 molecular mechanism. Upon TGFB1 stimulation, more circITGB6 are expressed to stabilize PDPN mRNA molecules through their interactions with IGF2BP3, thus facilitating EMT process and promoting tumor metastasis ²⁵.

Clinical Applications and Challenges.

Biomarkers
CircRNAs have recently emerged as promising cancer biomarkers and therapeutic targets due to their covalently closed structure, long half-life, and cell-type specificity ²⁹. These properties allow them to accumulate at high concentrations, remain stable, and be detectable in various body fluids—including plasma, urine, and saliva ³⁰. As a result, circRNAs are considered minimally invasive biomarkers for cancer diagnosis, particularly in diseases such as pancreatic ductal adenocarcinoma (PDAC) and glioblastoma, where late-stage detection is common ¹¹. Moreover, circRNA signatures derived from exosomes have been proposed to distinguish cancer stages and subtypes, for example in triple-negative breast cancer (TNBC) and glioblastoma, offering new diagnostic and prognostic tools ³¹.

Challenges of using circRNAs as biomarkers
Despite their potential, several challenges must be addressed before circRNAs can be fully integrated into clinical practice. Although circRNAs show cell-type–specific expression, an optimal biomarker must be truly cancer-specific to prevent false positives caused by signals from normal cells ³². Additionally, small or heterogeneous tumors may express circRNAs at levels too low to detect with standard methods like PCR, necessitating more sensitive and costly approaches such as circRNA sequencing ³³. To overcome these limitations, researchers are exploring tumor-derived exosome purification and advanced detection technologies. For instance, circEGFR has been identified as a bivalent circRNA functioning both as a biomarker and as a therapeutic target in TNBC through activation of autophagy ³⁴.

Therapeutic application
CircRNAs are also being investigated for therapeutic purposes. Developing circRNA-based therapies requires a deeper understanding of their molecular interactions and regulatory networks. Effective and safe therapeutic strategies must ensure durable responses, targeted delivery to cancer cells, and minimized pleiotropic effects ¹¹. CRISPR–Cas13 systems have been used to degrade circRNAs in cell lines and proposed for in vivo applications ³⁵, although Cas13’s nuclease activity lacks full specificity and may produce unintended effects ³⁶. Conversely, engineered circRNAs are being explored as delivery vehicles for therapeutic cargos, analogous to mRNA-based vaccines ³⁷.

Future Perspectives and Conclusions
CircRNAs contribute to multiple stages of oncogenesis, making the characterization of their stoichiometry, spatial localization, targets, and interactome essential for fully understanding their potential. Notably, some circRNAs physically interact with DNA to promote oncogenic mutations, suggesting a role in mutagenesis across several cancers, including acute leukemia. Their exceptional stability and detectability in liquid biopsies make circRNAs promising candidates for incorporation into cancer screening programs, particularly for tumors in hard-to-biopsy locations such as the brain. However, key challenges remain, including improving detection sensitivity for low-abundance, cancer-specific circRNAs and identifying circRNA panels suitable for screening. It also remains important to compare circRNA biomarkers with existing clinical markers in terms of specificity, sensitivity, and cost-effectiveness. CircRNAs function as both oncogenes and tumor suppressors in a cell type–specific manner, positioning them as attractive tools for personalized medicine with the potential to enhance treatment responses and patient survival. In the coming decade, circRNAs are expected to play an expanding role as therapeutic targets in vaccine delivery and gene therapy, though substantial investment is needed to optimize large-scale production, delivery methods, translation efficiency, and off-target risk mitigation. Beyond deepening our understanding of cancer biology, these advances may reveal new therapeutic targets. In particular, circITGB6 and its associated pathways may represent effective targets for inhibiting metastasis and improving colorectal cancer outcomes. Continued research is essential, especially efforts to identify all interactors of circITGB6, define its tissue-specific expression, and clarify its function across tumor types. As more circRNAs are linked to diverse cellular processes, their integration into personalized medicine and cancer screening programs may transform oncology and expand therapeutic possibilities for cancer patients.