Glycoproteogenomics characterizes the CD44 splicing code driving bladder cancer invasion

Bladder cancer (BC) management demands the introduction of novel molecular targets for precision medicine. Cell surface glycoprotein CD44 has been widely studied as a potential biomarker of BC aggressiveness and cancer stem cells. However, significant alternative splicing and multiple glycosylation generate a myriad of glycoproteoforms with potentially distinct functional roles. The lack of tools for precise molecular characterization has led to conflicting results, delaying clinical applications. Addressing these limitations, we have interrogated the transcriptome of a large BC patient cohort for splicing signatures. Remarkable CD44 heterogeneity was observed, as well as associations between short CD44 standard splicing isoform (CD44s), invasion and poor prognosis. In parallel, immunoassays showed that targeting short O-glycoforms could hold the key to improve CD44 cancer specificity. This prompted the development of a glycoproteogenomics approach, building on the integration of transcriptomics-customized datasets and glycomics for protein annotation from nanoLC-ESI-MS/MS experiments. The concept was applied to invasive human BC cell lines, glycoengineered cells, and tumor tissues, enabling unequivocal CD44s identification. Finally, we confirmed the link between CD44s and invasion in vitro by siRNA knockdown, supporting findings from BC tissues. The key role played by short-chain O-glycans in CD44-mediated invasion was also demonstrated through glycoengineered cell models. Overall, CD44s emerged as biomarker of poor prognosis and CD44-Tn/STn as promising molecular signatures for targeted interventions. This study materializes the concept of glycoproteogenomics and provides a key vision to address the cancer splicing code at the protein level, which may now be expanded to better understand CD44 functional role in health and disease.Significance StatementThe biological role of CD44, a cell membrane glycoprotein involved in most cancer hallmarks and widely explored in BC, is intimately linked to its protein isoforms. mRNA alternative splicing generates several closely related polypeptide sequences, which have so far been inferred from transcripts analysis, in the absence of workflows for unequivocal protein annotation. Dense O-glycosylation is also key for protein function and may exponentiate the number of proteoforms, rendering CD44 molecular characterization a daunting enterprise. Here, we integrated multiple molecular information (RNA, proteins, glycans) for definitive CD44 characterization by mass spectrometry, materializing the concept of glycoproteogenomics. BC specific glycoproteoforms linked to invasion have been identified, holding potential for precise cancer targeting. The approach may be transferable to other tumors, paving the way for precision oncology.