Universität zu Köln

Kirschberg, Matthias (2021). Reprogramming of skin keratinocytes by the E7 oncoprotein of HPV8. PhD thesis, Universität zu Köln.

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Abstract

The human papillomavirus type 8 (HPV8), an oncogenic member of the genus betapapillomavirus (betaHPV), is associated with the development of actinic keratoses and squamous cell carcinoma of the skin. The viral early proteins E6 and E7 are oncoproteins which are known to interact with important regulatory mechanisms of infected keratinocytes. However, the molecular details how this occurs in case of HPV8-E7 is still only poorly understood. Previous research of our group had already shown that HPV8-E7 plays a pivotal role in inducing and regulating keratinocyte proliferation and invasion. It was also shown that the extracellular matrix protein fibronectin and the integrin α3β1, expressed on HPV8 positive keratinocytes, is key in regulating the invasive behavior of these cells. Furthermore, we had shown that fibronectin is not only overexpressed in HPV8-E7 positive keratinocytes, but also found to be enriched in the peri-tumoral stroma of betaHPV-positive skin cancer. Therefore, the overall aim of this thesis was to arrive at a deeper understanding how HPV8-E7 reprograms the cell and how it hijacks vital cellular processes and how this interference may ultimately contribute to malignant transformation processes in HPV8 infected keratinocytes. To extend our knowledge of how these events occur transcriptomic, total and phospho-proteomics as well as protein-protein interaction assays were employed. cDNA microarray results led to the observation that global gene expression changes induced by HPV8-E7 are mainly accomplished through binding sites within the gene promoters for the transcription factors Sp1/Sp3. In transient transfection assays we could confirm that HPV8-E7 stimulates the activity of promoters of cellular genes which contain Sp1/Sp3 binding sites, including the fibronectin gene promoter. Interestingly the HPV8-E7L23A mutant, which is not able to trigger keratinocyte invasion was unable to activate the fibronectin promoter (Kirschberg*, Heuser* et al., 2019). By using the same cDNA microarray results GADD34 (growth arrest and DNA damage-inducible protein 34) and GDF15 (growth/differentiation factor 15) were identified as novel downstream genes targeted by E7. In addition, proteomic analyses of HPV8-E7 positive keratinocytes cultivated on a fibronectin matrix showed that the presence of E7 led to hyper- or hypo-phosphorylation of cellular proteins. Here, we also revealed that hypo-phosphorylated proteins are mainly associated with DNA damage repair and DNA replication, whereas hyper-phosphorylated proteins are apparently important for cytoskeletal organization and also include proteins associated with cell polarity. Therefore, these observations reveal that signals downstream of fibronectin resulting in hyper-phosphorylation of proteins are critical for cell invasion. Interestingly, among the identified up-stream kinases were the proto-oncogenic Src-kinase family members Lyn and Fyn, which are over-activated in the presence of HPV8-E7 (Kirschberg et al., 2021). Looking at direct E7 interacting proteins, NuMa (nuclear mitotic apparatus protein 1) and ATP5B (ATP synthase F1 subunit beta) were identified as novel HPV8-E7 interacting partners. E7 appears to co-localize with NuMa in mitotic cells. Curiously, in a small subset of these cells E7 was only found at one whereas NuMa was found at both spindle poles. This could implicate that the binding of E7 to NuMa at only one spindle pole might also affect the ratio of symmetric / asymmetric cell division of epidermal progenitor cells, which could explain the already known enrichment of stem-cell-like cancer cells in HPV8 positive primary keratinocyte cultures (Oswald*, Kirschberg* et al., 2019). The observed interaction of E7 and ATP5B was most intriguing as betaHPV E7 had been mainly known as a nuclear and cytoplasmic protein. The ability to bind ATP5B is not exclusive for HPV8 but could also be shown for low-risk (HPV11) and high-risk (HPV16) mucosa -infecting HPV. The association of E7 with ATP5B was accompanied by an increase in mitochondrial energy production. In particular E7 positive keratinocytes showed a stark increase in their spare respiratory capacity, allowing the cells to meet their heightened energy demands. The influence of ATP5B manipulation was further analyzed in oropharyngeal cancer patient groups which included both HPV16 positive and HPV-negative tumors. Interestingly, HPV-positive cancers showed a far more favorable outcome compared to HPV-negative tumors if ATP5B expression was high (Kirschberg et al., 2020). Having identified these new interaction partners, namely NuMa and ATP5B, it appears that E7 exerts its oncogenic functions in various cellular compartments. Virtually all so far published studies on the oncogenic functions of E7 were conducted in cells expressing the viral oncoproteins individually. Interestingly, when HPV8-E7 is co-expressed with HPV8-E6 other oncogenic functions appear to become active, leading to the CHK1 (checkpoint kinase-1) protein degradation. This disruption of CHK1 was mediated by manipulating the autophagic pathway that was so far not known to be a degradation pathway used by HPV oncoproteins (Akgül, Kirschberg et al., 2019).

Item Type:	Thesis (PhD thesis)
Translated title:	Title Language Reprogrammierung von Hautkeratinozyten durch das E7 Onkoprotein von HPV8 German
Creators:	Creators Email ORCID ORCID Put Code Kirschberg, Matthias matthias.kirschberg@yahoo.com UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-533235
Date:	7 September 2021
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Medicine > Virologie > Institut für Virologie
Subjects:	Life sciences
Uncontrolled Keywords:	Keywords Language betapapillomavirus, Dermato-Oncology, Proteomics, Metabolics, Transcriptomics English
Date of oral exam:	8 June 2021
Referee:	Name Academic Title Akgül, Baki Univ.-Prof. Dr. Björn, Schumacher Univ.-Prof. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/53323