Universität zu Köln

Balaji, Harini (2026). Analysis of oxidative stress and metabolic reprogramming in HPV+ HNSCC. PhD thesis, Universität zu Köln.

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Abstract

HPV-positive (HPV⁺) oropharyngeal squamous cell carcinoma (OPSCC) represents a biologically distinct subset of head and neck cancer with generally favourable outcomes compared to HPV-negative (HPV-) disease. Nevertheless, 20–25% of HPV⁺ OPSCC patients develop recurrence or metastasis, reflecting a clinically relevant subgroup with poor prognosis. This aggressive behaviour is frequently associated with HPV16 host genome integration and high expression of the splice variant HPV16-E6*I, which often co-occurs with PI3K pathway mutations and 3q chromosomal gains. These tumours adopt metabolic and redox profiles resembling HPV⁻ cancers, illustrating the limitations of current p16INK4a-based risk stratification and underscoring the need for HPV-specific biomarkers to guide therapy. This builds on previous research from our group, which identified that HPV16 integration and HPV16-E6*I overexpression and are both associated with unfavourable disease outcome. To address this, we developed FFPE-targeted locus capture (FFPE-TLC), a proximity-ligation sequencing method optimised for formalin-fixed, paraffin-embedded tissue. FFPE-TLC achieves high-resolution mapping of virus–host breakpoints and local structural variation, enabling integration analysis directly from diagnostic samples. In a cohort of HPV16⁺ OPSCC, ~56% of tumours contained HPV integration, frequently accompanied by simple rearrangements and intratumour heterogeneity. Breakpoints often localised near genes involved in DNA repair, epithelial–mesenchymal transition (EMT), and apoptosis signalling. Importantly, shared integration sites in primary and metastatic lesions confirmed viral insertion as a stable clonal marker. To functionally link integration-associated isoforms with phenotype, we established HEK 293 models stably overexpressing HPV16-E6 or -E6*I under O₂ levels that mimic tonsillar physiology. HPV16-E6*I broadly induced NRF2 and AKR1C3 expression with O₂-sensitive differences in subcellular localisation. Functionally, HPV16-E6*I enhanced migration under both hypoxia and hyperoxia and promoted a dual-fuel phenotype: oxidative phosphorylation (OXPHOS) activation with ATP advantage at high O₂, and glycolytic flexibility under low O₂. By contrast, HPV16-E6 favoured glycolysis at physiologic normoxia. Inhibition of AKR1C1/3 reversed transcript changes and abolished migration, highlighting these enzymes as critical effectors of the HPV16-E6*I-driven redox–motility axis. For translational application, we established isoform-specific RNA-FISH using locked nucleic acid (LNA) probes for HPV16-E6 and -E6*I in FFPE material. This enables spatial mapping of isoform expression in situ, which can be combined with FFPE-TLC data to stratify tumours by integration status, redox activity, and metabolic adaptation. In conclusion, our study defines HPV16-E6*I as a central regulator linking viral integration to O₂-sensitive redox and metabolic reprogramming, thereby driving aggressive tumour behaviour in a clinically relevant OPSCC subgroup. By introducing FFPE-TLC and isoform-specific RNA-FISH as robust, clinically applicable tools, we provide a framework for biomarker-driven risk stratification and open new perspectives for tailored therapeutic strategies beyond p16-based classification.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Balaji, Harini harini.balaji@uk-koeln.de UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-793247
Date:	2026
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Medicine > Hals-Nasen-Ohrenheilkunde > Klinik und Poliklinik für Hals-, Nasen- und Ohrenheilkunde
Subjects:	Natural sciences and mathematics
Uncontrolled Keywords:	Keywords Language HPV+ UNSPECIFIED HNSCC UNSPECIFIED oxidative stress UNSPECIFIED metabolic reprogramming UNSPECIFIED viral integration UNSPECIFIED
Date of oral exam:	22 October 2025
Referee:	Name Academic Title Klußmann, Jens Peter Prof. Dr. Schumacher, Björn Prof. Dr. Lemberg, Marius Prof. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/79324