The human transcriptome contains thousands of small open reading frames (sORFs) that encode microproteins whose functions remain largely unexplored. Here, we show that TINCR lncRNA encodes pTINCR, an evolutionary conserved ubiquitin-like protein (UBL) expressed in many epithelia and upregulated upon differentiation and under cellular stress. By gain- and loss-of-function studies, we demonstrate that pTINCR is a key inducer of epithelial differentiation in vitro and in vivo. Interestingly, low expression of TINCR associates with worse prognosis in several epithelial cancers, and pTINCR overexpression reduces malignancy in patient-derived xenografts. At the molecular level, pTINCR binds to SUMO through its SUMO interacting motif (SIM) and to CDC42, a Rho-GTPase critical for actin cytoskeleton remodeling and epithelial differentiation. Moreover, pTINCR increases CDC42 SUMOylation and promotes its activation, triggering a pro-differentiation cascade. Our findings suggest that the microproteome is a source of new regulators of cell identity relevant for cancer.

Recent work has unveiled a hidden microproteome composed by thousands of small proteins named microproteins: they are functional short proteins coded by genomic regions previously considered non-coding, and which had been completely ignored, mainly due to their small size (<100 aminoacids). To date, only a small part of the thousands of microproteins present in our cells have been characterized, and they are key players in fundamental processes such as DNA repair, mRNA splicing or cell metabolism. In cancer, they have been shown to regulate most tumor hallmarks, and present a huge potential for the clinic as diagnostic and prognostic biomarkers as well as therapeutic targets. Interestingly, their small size makes them ideal candidates to be shed in tumor-derived exosomes. PDAC-shed exosomes have been shown to prepare the pre-metastatic niche in the liver, and their presence in the bloodstream can be used as a surrogate marker of metastasis. Herein, we have mined the PDAC exosome-secreted microproteome for novel regulators of tumor progression and metastasis. Using proteogenomics in PDAC patient-derived explants, we have identified 439 microproteins secreted in exosomes by pancreatic tumors. We have selected a set of top microprotein candidates for further characterization by in silico analyses (e.g. phylogenetic conservation, predicted protein stability and localisation, mRNA expression in PDAC, etc). We have confirmed their exosome secretion in PDAC cell lines, and preliminary characterisation of these top candidates has shown that they extrinsically promote PDAC cell growth and invasion in vitro. Together, this work advances our knowledge on the underexplored field of secreted microproteins and provides pioneering evidence of their role in tumor cell communication in PDAC. It may further be a source of novel therapeutic targets and PDAC biomarkers for liquid biopsy in the clinic. Citation Format: Marion Martinez, Marta Hergueta, Pilar Ximénez de Embún, Ana Dueso, David Torrents, Teresa Macarulla, Javier Muñoz, Héctor Peinado, María Abad. Mining the secreted microproteome for novel regulators of PDAC progression [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C074.