Abstract 6964: Regulation of TGF-β signaling via a novel TβRIII sheddase

Abstract

Abstract In a normal cell, TGF-β signaling serves as a tumor suppressor by reducing proliferation and promoting apoptosis. Paradoxically, the function of TGF-β signaling reverses in a cancer cell and begins to promote oncogenic pathways such as proliferation, survival, evasion of the immune system, and epithelial to mesenchymal transition (EMT). This has made TGF-β signaling a very attractive therapeutic target for a wide variety of human carcinomas. As a result, many small molecule inhibitors of TGF-β signaling have been developed, but none have achieved FDA approval for use in human cancers due to the deleterious effects of inhibiting TGF-β responsiveness in normal cells. In effort to overcome this obstacle, we aim to restore the normal biological mechanisms that regulate TGF-β signaling. Specifically, the membrane bound co-receptor TβRIII can be proteolytically cleaved and shed from the membrane, resulting in an extracellular protein capable of binding and sequestering TGF-β ligand. This decoupling of ligand binding from the receptor allows TβRIII to suppress signaling. TβRIII shedding decreases TGF-β mediated oncogenic phenotypes such as migration and metastasis in multiple cancer types. However, this shedding is often lost in cancer. Therefore, restoration of TβRIII shedding could be a viable therapeutic approach for multiple cancer types. One potential mechanism for the loss of shedding is the negative regulation of the protease responsible for shedding (sheddase). However, the identity of the sheddase still remains unknown. Here, our goal is to identify the sheddase and uncover regulatory mechanisms that could be leveraged to restore TβRIII shedding in cancer. To achieve this goal, a library of protease CRISPR knockout viruses was screened to determine which proteases have an effect on shedding. The quantity of TβRIII shed into the cell culture media was measured via ELISA for each of these CRISPR knockout cell lines, and the candidates with the largest reduction of shedding were retained for further investigation. The list of candidates was further screened in silico via gene set enrichment analysis of human patient data against a TGF-β signaling gene set signature. Only the candidates demonstrating a negative correlation between sheddase candidate expression and TGF-β signaling activity were studied further. To further validate these candidate sheddases, knockdown and overexpression cell lines were created and characterized. Overexpression of the most promising candidate reduced TGF-β mediated phenotypes such as migration and invasion, while knockdown of this candidate increased the same phenotypes. Knockdown also increased expression of TGF-β and EMT markers. Importantly, these effects of sheddase knockdown were neutralized in the presence of the TGF-β inhibitor galunisertib or when the cells were rescued with exposure to purified soluble TβRIII, indicating the effects are mediated through TβRIII shedding. Citation Format: Benjamin M. Greulich, John Pawlak, John Post, Elena Karas, Gerard Blobe. Regulation of TGF-β signaling via a novel TβRIII sheddase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6964.