Molecular Cancer | circNSD2 Promotes Triple-Negative Breast Cancer Metastasis and Immune Escape via the USP10/SRSF6/TPM1 Axis

This study systematically reveals the molecular mechanism by which the TGF-β signaling pathway regulates circRNA, providing new intervention targets for metastasis and immune escape in TNBC. It suggests that combined targeting of circNSD2 and PD-L1 may enhance anti-tumor immune responses.

 

Literature Overview

This article, 'CircNSD2 promotes metastasis and immune escape by increasing the USP10/SRSF6-mediated alternative splicing of TPM1 in TNBC,' published in the journal Molecular Cancer, systematically investigates how TGF-β signaling drives invasion, metastasis, and immune escape in triple-negative breast cancer (TNBC) by inducing circNSD2 expression. By integrating high-throughput sequencing, functional experiments, and clinical data, the study reveals a novel scaffolding role of circNSD2 as an RNA molecule, expanding our understanding of non-coding RNAs in the regulation of the tumor microenvironment.

Background Knowledge

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer due to the lack of ER, PR, and HER2 expression, resulting in limited treatment options and a high propensity for distant metastasis. The TGF-β signaling pathway plays a dual role in TNBC: tumor-suppressive in early stages and tumor-promoting in later stages, particularly by driving epithelial–mesenchymal transition (EMT) and metastasis. However, its downstream effectors remain incompletely defined. In recent years, circRNAs have emerged as potential biomarkers and therapeutic targets due to their high stability and tissue specificity. Nevertheless, how circRNAs are regulated by TGF-β and whether they participate in immune checkpoint regulation, such as PD-L1 expression, remain major research challenges. This study initiates from screening TGF-β-responsive circRNAs, focusing on circNSD2, and systematically elucidates its functional and mechanistic roles in TNBC progression, thereby filling a critical knowledge gap in the TGF-β–circRNA–immune escape axis.

 

 

Research Methods and Experiments

The study utilized TNBC cell lines MDA-MB-231 and MDA-MB-468, combined with TGF-β treatment and high-throughput circRNA sequencing, to identify differentially expressed circRNAs. Loss-of-function and gain-of-function experiments, along with Transwell assays, 3D tumor spheroid invasion, and mouse lung metastasis models, were employed to validate the impact of circNSD2 on tumor cell migration, invasion, and colonization capabilities in vitro and in vivo. Mechanistically, ChIP, luciferase reporter assays, and RNA pulldown coupled with mass spectrometry were used to systematically dissect the transcriptional regulation and protein interaction network of circNSD2. Clinical relevance was assessed by qRT-PCR, ISH, and IHC in patient cohorts to correlate circNSD2 expression with metastasis and prognosis.

Key Conclusions and Perspectives

• TGF-β activation enables SMAD2 to bind the NSD2 promoter region, directly promoting circNSD2 transcription. This reveals a transcriptional regulatory mechanism of TGF-β on circRNA and provides a paradigm for studying other TGF-β-responsive circRNAs.
• circNSD2 circularization depends on the RNA-binding protein KHSRP, which is itself regulated by TGF-β, forming a positive feedback loop. This suggests KHSRP as a potential target for interfering with circNSD2 biogenesis.
• circNSD2 acts as a molecular scaffold to facilitate the interaction between SRSF6 and USP10, inhibiting K48-linked ubiquitination and degradation of SRSF6, thereby stabilizing the SRSF6 protein. This mechanism expands the functional model of circRNAs as protein interaction platforms and offers new insights into targeting protein stability.
• The stabilized SRSF6 regulates alternative splicing of TPM1, promoting an invasive phenotype in tumor cells. This reveals the role of the circRNA–splicing factor–effector gene axis in EMT and suggests TPM1 isoforms as molecular markers for TNBC progression.
• circNSD2 upregulates PD-L1 expression and suppresses CD8+ T cell anti-tumor functions, directly linking circRNA to immune checkpoint pathways. This supports the potential of circNSD2 as a predictive biomarker for immunotherapy sensitivity.

Research Significance and Prospects

This study provides a dual intervention strategy for precision therapy in TNBC: on one hand, targeting circNSD2 or its interacting proteins USP10/SRSF6 could inhibit tumor metastasis; on the other, combining anti-PD-L1 therapy may reverse immune escape and improve therapeutic efficacy. Moreover, the high stability and specificity of circNSD2 make it a promising non-invasive biomarker in liquid biopsies for monitoring metastatic risk and treatment response in TNBC patients.

 

 

Conclusion

This study systematically elucidates the central role of the TGF-β–circNSD2–USP10/SRSF6/TPM1–PD-L1 signaling axis in TNBC metastasis and immune escape, extending the functional scope of circRNAs from 'miRNA sponges' to 'protein scaffolds,' and for the first time directly linking them to alternative splicing and immune checkpoint regulation. This discovery not only deepens our understanding of the mechanisms underlying TNBC malignancy but also provides a translational pathway from molecular diagnostics to combination therapy. In the future, developing ASOs or small-molecule inhibitors targeting circNSD2, in combination with immune checkpoint inhibitors, may become a key clinical strategy for TNBC, advancing treatment paradigms from 'single-target' to 'network intervention' and laying a solid foundation for improving patient outcomes.

 

Yuhan Sun, Yaming Li, Tong Niu, Xiaoli Kong, and Qifeng Yang. CircNSD2 promotes metastasis and immune escape by increasing the USP10/SRSF6-mediated alternative splicing of TPM1 in TNBC. Molecular Cancer.