Elucidating the Mechanisms of HPV-Induced Breast Cancer

Abstract

Human papillomavirus (HPV) is a well-established oncovirus responsible for several human tumours, including cervical, head and neck, and penile cancers. Its oncoproteins, E6 and E7, are central to oncogenesis through diverse mechanisms including the inactivation of p53 and pRb, consequently implicating in deregulated cell proliferation and immune evasion. Although, recent studies have detected HPV DNA and, in some cases, its oncoproteins in breast tumours, the mechanistic role of HPV in breast carcinogenesis remains poorly understood. Clarifying whether HPV contributes actively to breast cancer initiation or progression is essential for informing targeted prevention and therapeutic interventions. This study aimed to elucidate the mechanisms by which HPV contributes to breast cancer development or progression through the expression and activities of its E6 and E7 oncoproteins. Breast tumour and matched adjacent non-neoplastic tissue samples previously screened for HPV DNA by PCR were analysed for E6 and E7 protein expression using enzyme-linked immunosorbent assay (ELISA). Functional assays were then performed using the MDA-MB-468 breast cancer cell-line transfected with HPV E6/E7 plasmids to assess their effects on migration and immune-related pathways. Wound-healing assays were conducted to evaluate cell motility, while qRT-PCR assessed the expression of migration (ZEB1, VIM, CDH1, CDH2, MMP1, SNAIL) and immune-related (VEGF, IL6, IL8, CXCR4) genes. Additionally, in silico analyses using 60 RNA-Seq datasets from HPV-positive and HPV-negative oropharyngeal, head and neck, and vulvar cancers were performed to identify shared molecular drivers (hub genes) through Weighted Gene Co-expression Network Analysis (WGCNA). These hub genes were further validated by LASSO regression as strong predictors of HPV-associated cancers tissue types. ELISA results revealed E6 and E7 oncoproteins were expressed in HPV-positive breast tumours compared with HPV-negative and adjacent tissues (p < 0.01), indicating active viral expression. Functionally, E6/E7-transfected MDA-MB-468 cells exhibited enhanced directional migration, with nearly complete wound closure by 60 hours, compared with minimal closure in controls. Gene expression analyses revealed significant upregulation of CDH2, MMP1, and SNAIL (p < 0.01) alongside reduced CDH1 expression (p < 0.001), suggesting induction of partial epithelial–mesenchymal transition. In co-culture assays with peripheral blood mononuclear cells (PBMCs), IL6 and CXCR4 were significantly upregulated (p < 0.01), whereas VEGF remained unchanged, indicating selective activation of inflammatory and chemotactic pathways. Bioinformatic analysis revealed distinct HPV-associated signatures. HPV-positive tumours showed upregulation of immune-related genes (CD3E, CD4, CD8A, CD2, CD27) associated with enhanced T-cell activation and tumour infiltration, whereas HPV-negative tumours were enriched for extracellular matrix genes (FN1, COL6A1, COL1A1, COL1A2) linked to stromal stiffness. These findings suggest that HPV-positive tumours display an immune-inflamed but less fibrotic microenvironment compared with HPV-negative cases. The study provides molecular and functional evidence that HPV, through its E6 and E7 oncoproteins, may contribute to breast tumour biology beyond incidental presence. Elevated viral protein expression, enhanced cell migration, induction of pro-inflammatory genes, and activation of shared molecular networks across HPV associated cancers collectively suggest biologically active viral involvement in breast carcinogenesis. These findings strengthen the need for further and better models such as longitudinal studies to confirm HPV’s causal role and its potential relevance to breast cancer prevention strategies.