Cancers driven by disruption of the retinoblastoma (RB) tumour suppressor pathway are still frequently detected at advanced stages, despite long-standing molecular insight into their biology. In high-risk human papillomavirus (HPV) associated cancers, the E7 oncoprotein promotes oncogenesis by targeting RB for degradation, resulting in loss of cell cycle control. Although preventive vaccination and screening have reduced disease burden in some populations, substantial gaps remain in early detection and risk stratification, particularly for HPV-driven and RB-dysregulated malignancies. Identifying biomarkers that directly reflect core oncogenic mechanisms and show consistency across tumour types is therefore a critical priority for cancer prevention and emerging multi-cancer early detection (MCED) strategies.

To identify novel cellular regulators of HPV E7 function, characterise their role in RB pathway stability, and evaluate their potential as translational biomarkers relevant to early cancer detection and prevention.

A Gal4 yeast two-hybrid screen was employed to identify cellular interactors of high-risk HPV E7. Functional characterisation was performed using transient and stable expression systems across HPV positive (HeLa, SiHa), HPV-negative (C33A), and additional epithelial and control cell models. Protein expression, stability, and molecular interactions were assessed by Western blotting using antibodies against E7, RB, and the candidate protein, secreted novel tumour suppressor protein (sNTSP). Cellular proliferation assays were used to evaluate biological effects. Extracellular secretion of sNTSP was examined through conditioned media analyses. Translational relevance was investigated through immunohistochemical and immunoflourescent assessment of sNTSP expression across multiple tumour types with matched normal tissues, with quantitative digital pathology analyses.

sNTSP was identified as a strong and reproducible interactor of high-risk HPV E7. Co-expression of sNTSP antagonised E7-mediated oncogenic phenotypes, reversing E7-induced cellular proliferation and protecting RB from degradation. sNTSP increased RB protein abundance and prolonged its half life in a dose dependent manner. Significantly, it was discovered that sNTSP was secreted via non-classical pathway and maintained tumour suppressor activity in neighbouring cells, stabilising RB through a previously unrecognised paracrine mechanism. Multiplexed immunofluorescence and Confocal microscopy showed RB, sNTSP and E7 colocalize inside the cells. Furthermore, Immunohistochemical analyses shows a consistent reduction of sNTSP expression in tumour tissues when compared to corresponding normal tissues across multiple cancer types, suggesting that loss of sNTSP may represent a conserved feature of malignancy.

This study identifies that sNTSP is a novel secreted tumour suppressor that counteracts HPV E7 mediated RB degradation via paracrine mechanisms. The consistent downregulation of sNTSP across diverse malignancies highlights its potential utility as a cross-cancer biomarker that reflects RB pathway disruption. Our work will provide scientific evidence for incorporating sNTSP into future biomarker panels and MCED frameworks, supporting earlier diagnosis and strengthening cancer prevention efforts in line with the mission of the International Agency for Research on Cancer.