Illuminating the Shadows: Functional Roles of the Dark Proteome in Cancer Progression

Michel Salzet
Univ.Lille, Inserm, CHU Lille, U1192, Laboratoire Protéomique, Réponse Inflammatoire et Spectrométrie de Masse (PRISM), F-59000 Lille, France

Michel Salzet, PhD (Presenter) Laboratoire PRISM - Université de Lille

Presenter Bio: Professor Michel salzet (Distinguish Professor) Is a member of the Institut Universitaire de France in the speciality of immunoproteomic. He is director of the unit, UMR 1192 Inserm. He discovered with Professor Xavier Roucou, the ghost proteins through proteomic approaches combined with Bioinformatic analyses. For this achievement, they received the Quebec science Award for the discovery of the year 2014. Professor Michel Salzet has (co-)authored more than 437 papers (H-index 71, google scholar) and he has published in Top notch journals like Cancer Cells, Nature protocols, Nature Immunology, Circulation, Blood, Trends in immunology.

Relevant Financial Disclosures (within past 24 months, reported on May 15, 2025) No relevant financial relationship(s) to disclose.

INTRODUCTION
The classical perception of the eukaryotic proteome has been radically transformed with the recognition that large genomic regions previously categorized as non-coding DNA encode functional peptides, now termed the "dark proteome" (1-3). These noncanonical proteins, often under 100 amino acids, arise from alternative open reading frames (altORFs), small ORFs (smORFs), untranslated regions (UTRs), and noncoding RNAs (ncRNAs). Their identification and validation have been made possible through technological advances in proteogenomics, notably ribosome profiling (4), deep high-resolution mass spectrometry (MS), and sophisticated bioinformatics. The dark proteome has emerged as a major player in cancer biology, revealing unappreciated layers of regulatory control over oncogenesis.

METHODS
The study of the dark proteome employs a multidisciplinary approach combining state-of-the-art proteogenomics pipelines. Ribosome profiling is used to detect active translation events across noncanonical coding regions (4). Deep MS and advanced crosslinking mass spectrometry (XL-MS) facilitate the identification and mapping of protein–protein interaction networks (5,6). Bioinformatic analysis incorporating evolutionary conservation metrics distinguishes functional peptides from noise. In parallel, machine learning-driven rescoring strategies improve sensitivity and specificity of dark proteome identification from complex MS datasets (1).

RESULTS
Characterization of alternative proteins has revealed their key roles in processes such as cell proliferation, apoptosis, metabolic reprogramming, immune evasion, and genomic stability (7-10). Notable examples include AltAKT and AltEDARADD, which modulate cancer cell proliferation; HOXB-AS3, which acts as a tumor suppressor by destabilizing oncogenic mRNAs (3,9) and EMBOW, which regulates cell cycle progression via interactions with chromatin modifiers. These microproteins integrate into classical signaling cascades and often function as modulators or gatekeepers of critical oncogenic pathways. Evolutionary analyses reveal both recently emerged, species-specific proteins and ancient conserved microproteins, underscoring their diverse functional relevance. Moreover, their tumor-specific expression and small size present promising opportunities for use as neoantigen sources in cancer immunotherapy and as scaffolds for drug design (1).

CONCLUSION
The systematic exploration of the dark proteome is redefining the molecular landscape of cancer research. While challenges remain in distinguishing biologically relevant alternative proteins from transient translation products, integrative multi-omic approaches and continual improvements in detection and functional annotation methods are driving progress. These efforts are poised to expand the repertoire of cancer biomarkers and therapeutic targets. Ultimately, unveiling these hidden proteins may deliver transformative insights into oncogenesis and precision oncology, offering novel avenues for diagnosis, prognosis, and treatment.

REFERENCES

1. Orr MW, Mao Y, Storz G, Qian SB. Alternative ORFs and small ORFs: shedding light on the dark proteome. Nucleic Acids Res. 2020;48(3):1029-1042.
2. Vanderperre B, Lucier J-F, Bissonnette C, et al. Direct detection of alternative open reading frames translation products in human significantly expands the proteome. PLoS One. 2013;8(8):e70698.
3. Cardon T, Fournier I, Salzet M. Shedding light on the ghost proteome. rends in Biochemical Sciences 2021; 46.3: 239-250.
4. Ingolia NT, Lareau LF, Weissman JS. Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell. 2011;147(4):789-802.
5. Cardon T, Franck J, Coyaud E, Laurent EMN, Damato M, Maffia M, Vergara D, Fournier I, Salzet M. Alternative proteins are functional regulators in cell reprogramming by PKA activation. Nucleic Acids Res. 2022, 48(14):7864-7882.
6. Garcia-Del Rio DF, Derhourhi M, Bonnefond A, Leblanc S, Guilloy N, Roucou X, Eyckerman S, Gevaert K, Salzet M, Cardon T. Deciphering the ghost proteome in ovarian cancer cells by deep proteogenomic characterization. Cell Death Dis. 2024; 15(9):712
7. Duhamel M, Drelich L, Wisztorski M, Aboulouard S, Gimeno JP, Ogrinc N, Devos P, Cardon T, Weller M, Escande F, Zairi F, Maurage CA, Le Rhun É, Fournier I, Salzet M. Spatial analysis of the glioblastoma proteome reveals specific molecular signatures and markers of survival. Nat Commun. 2022;13(1):6665.
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9. Huang JZ, Chen M, Chen D, et al. A Peptide Encoded by a Putative lncRNA HOXB-AS3 Suppresses Colon Cancer Growth. Mol Cell. 2017;68(1):171-184.e6.
10. Aboulouard S, Wisztorski M, Duhamel M, Saudemont P, Cardon T, Narducci F, Lemaire AS, Kobeissy F, Leblanc E, Fournier I, Salzet M. In-depth proteomics analysis of sentinel lymph nodes from individuals with endometrial cancer. Cell Rep Med. 2021;2(6):100318.