Most colorectal cancer (CRC)-related deaths are associated to metastasis, with the liver being the primary organ for CRC colonization. Due to the lack of efficient treatments for cancer metastasis, the identification of new therapeutic targets either direct or upstream regulators of selected targets that focus on this metastasis is crucial to reduce CRC mortality. Tropomodulin-2 (TMOD2), a protein not previously linked to CRC, was found upregulated in highly metastatic to liver CRC cells. Here, we aimed to functionally characterize TMOD2"s role, and its interactome, and proteome in CRC.

Methods involved inducing stable TMOD2 overexpression and transient silencing in low-metastatic and highly metastatic isogenic CRC cell models. In vitro and in vivo functional assays were performed to determine TMOD2"s role in CRC cells" tumorigenic and metastatic properties. Furthermore, a quantitative label free (LFQ) proteomics approach was performed to characterize the differential proteome associated with TMOD2 overexpression using an Orbitrap Astral mass spectrometer. In addition, an immunoprecipitation (IP) coupled with LC-MS/MS was performed to characterize the interactome associated with TMOD2 using a Q Exactive mass spectrometer..

Results suggested an important role of TMOD2 in inducing proliferation, migration, and anchorage-independent growth capacities of CRC cells. Moreover, TMOD2 was implicated in improving liver colonization and tumoral growth in metastatic niches and was observed to be involved in the regulation of focal adhesions. Quantitative proteomics identified 350 out of 5540 proteins as significantly dysregulated upon TMOD2 overexpression, with selected proteins whose dysregulation was confirmed by WB. By IP, 115 potential TMOD2 interacting proteins were identified, and 13 were validated as actual interactors by WB and immunofluorescence.

These findings suggest a novel role of TMOD2 in CRC progression and metastasis, linking it to the induction of CRC cell migration, liver colonization, and proliferation. These enhanced capabilities characterized by functional proteomics may be associated with the increased number of focal adhesions induced by TMOD2 upregulation, and the differential proteome and interactome associated with TMOD2.