Simple Summary Lately, human cytomegalovirus (HCMV) has been progressively implicated in carcinogenesis alongside its oncomodulatory impact. CMV-Transformed Human mammary epithelial cells (CTH) phenotype might be defined by giant cell cycling, whereby the generation of polyploid giant cancer cells (PGCCs) could expedite the acquisition of malignant phenotypes. Herein, the main study objectives were to assess the transformation potential in vitro and evaluate the obtained cellular phenotype, the genetic and molecular features, and the activation of cellular stemness programs of HCMV strains, B544 and B693, which were previously isolated from triple-negative breast cancer (TNBC) biopsies. The strains' sensitivity to paclitaxel and ganciclovir combination therapy was evaluated. A unique molecular landscape was unveiled in the tumor microenvironment of TNBC harboring high-risk HCMV. Overall, the explicit oncogenic and stemness signatures highlight HCMV potential in breast cancer progression thus paving the way for targeted therapies and clinical interventions which prolong the overall survival of breast cancer patients. Background: Human cytomegalovirus (HCMV) oncomodulation, molecular mechanisms, and ability to support polyploid giant cancer cells (PGCCs) generation might underscore its contribution to oncogenesis, especially breast cancers. The heterogeneity of strains can be linked to distinct properties influencing the virus-transforming potential, cancer types induced, and patient's clinical outcomes. Methods: We evaluated the transforming potential in vitro and assessed the acquired cellular phenotype, genetic and molecular features, and stimulation of stemness of HCMV strains, B544 and B693, isolated from EZH2(High)Myc(High) triple-negative breast cancer (TNBC) biopsies. Therapeutic response assessment after paclitaxel (PTX) and ganciclovir (GCV) treatment was conducted in addition to the molecular characterization of the tumor microenvironment (TME). Findings: HCMV-B544 and B693 transformed human mammary epithelial cells (HMECs). We detected multinucleated and lipid droplet-filled PGCCs harboring HCMV. Colony formation was detected and Myc was overexpressed in CMV-Transformed-HMECs (CTH cells). CTH-B544 and B693 stimulated stemness and established an epithelial/mesenchymal hybrid state. HCMV-IE1 was detected in CTH long-term cultures indicating a sustained viral replication. Biopsy B693 unveiled a tumor signature predicting a poor prognosis. CTH-B544 cells were shown to be more sensitive to PTX/GCV therapy. Conclusion: The oncogenic and stemness signatures of HCMV strains accentuate the oncogenic potential of HCMV in breast cancer progression thereby leading the way for targeted therapies and innovative clinical interventions that will improve the overall survival of breast cancer patients.
