Cleavage of tropomodulin-3 by asparagine endopeptidase promotes cancer malignancy by actin remodeling and SND1/RhoA signaling

Background Abnormal proliferation and migration of cells are hallmarks of cancer initiation and malignancy. Asparagine endopeptidase (AEP) has specific substrate cleavage ability and plays a pro-cancer role in a variety of cancers. However, the underlying mechanism of AEP in cancer proliferation and migration still remains unclear. Methods Co-immunoprecipitation and following mass spectrometry were used to identify the substrate of AEP. Western blotting was applied to measure the expression of proteins. Single cell/nuclear-sequences were done to detect the heterogeneous expression of Tmod3 in tumor tissues. CCK-8 assay, flow cytometry assays, colony formation assay, Transwell assay and scratch wound-healing assay were performed as cellular functional experiments. Mouse intracranial xenograft tumors were studied in in vivo experiments. Results Here we showed that AEP cleaved a ubiquitous cytoskeleton regulatory protein, tropomodulin-3 (Tmod3) at asparagine 157 (N157) and produced two functional truncations (tTmod3-N and tTmod3-C). Truncated Tmod3 was detected in diverse tumors and was found to be associated with poor prognosis of high-grade glioma. Functional studies showed that tTmod3-N and tTmod3-C enhanced cancer cell migration and proliferation, respectively. Animal models further revealed the tumor-promoting effects of AEP truncated Tmod3 in vivo. Mechanistically, tTmod3-N was enriched in the cell cortex and competitively inhibited the pointed-end capping effect of wild-type Tmod3 on filamentous actin (F-actin), leading to actin remodeling. tTmod3-C translocated to the nucleus, where it interacted with Staphylococcal Nuclease And Tudor Domain Containing 1 (SND1), facilitating the transcription of Ras Homolog Family Member A/Cyclin Dependent Kinases (RhoA/CDKs). Conclusion The newly identified AEP-Tmod3 protease signaling axis is a novel dual-regulation mechanism of tumor cell proliferation and migration. Our work provides new clues to the underlying mechanisms of cancer proliferation and invasive progression and evidence for targeting AEP or Tmod3 for therapy.

Automated summary

The AEP-Tmod3 protease signaling axis identified in this study is a novel dual-regulation mechanism of tumor cell proliferation and migration. The cleavage of Tmod3 by AEP generates truncated forms tTmod3-N and tTmod3-C, which enhance cancer cell migration and proliferation, respectively. Functional experiments and animal models confirmed the tumor-promoting effects of AEP truncated Tmod3, providing new insights for targeting AEP or Tmod3 for therapy.