Dissecting the transcriptome of CD44 in triple negative breast cancer

The CD44 receptor is a transmembrane glycoprotein involved in the recognition of specific extracellular matrix (ECM) ligands. It has a complex transcriptional regulation characterized by different splicing variants, many of which play a role in promoting tumorigenesis. Therefore, CD44 variants may serve as prognostic biomarkers and therapeutic targets. Triple-negative breast cancer (TNBC) is a poor prognosis subtype of breast adenocarcinomas characterized by the lack of druggable molecular hallmarks. TNBC represents a prototypical model for studying CD44's pro-tumoral activity. However, the regulation of the CD44 transcriptome and its influence on TNBC aggressiveness remains elusive.In this study, we dissected the CD44 transcriptome in the TNBC cell lines MDA-MB-231 and MDA-MB-468, which exhibit different malignant properties. MDA-MB-231 cells display higher extracellular matrix invasive potential, more prominent metastatic potential, and poorer response to chemotherapy compared to MDA-MB-468 cells. Finally, we induced the overexpression of a differentially expressed CD44 isoform between the two cell lines using transfection assays. We then performed whole transcriptome RNA-sequencing analysis of the transfected cells and their control. We identified distinct patterns in the CD44 transcriptome between the two TNBC cell lines. Specifically, among the top 5 differentially expressed (DE) CD44 isoforms compared to the control cell line, we detected the predicted transcript variant XM_017018585.2. This variant will be further confirmed through PCR and Sanger sequencing assays. To functionally characterize XM_017018585.2, we performed overexpression of the exogenous transcript in the MDA-MB-468 cell line using viral transfection. The overexpression of XM_017018585.2 induced upregulation of transcripts involved in the MAPK8 signaling pathway and downregulation of interferon response programs. Interestingly, the same downregulated genes were also observed in the estrogen-responsive breast cancer cell line MCF-7 upon silencing of specific pathways.The CD44 transcriptome analysis of TNBC cell models revealed novel transcriptional variants with potential significance in the modulation of the malignant phenotype. This study could shed light on the role of previously unidentified CD44 isoforms in promoting tumorigenesis. Furthermore, it could uncover new candidate diagnostic and prognostic biomarkers based on CD44 variant expression.