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Skin Keratinocyte Stem CEll proliferatioN in field CancErisation

Periodic Reporting for period 1 - SKin SCiENCE (Skin Keratinocyte Stem CEll proliferatioN in field CancErisation)

Reporting period: 2018-06-01 to 2020-05-31

The objective of the action was to strength the idea that β-HPV reactivation is involved in the development of SCC and finding the molecular mechanism that drives it. In immune compromised patients, the cancer that occurs can be due to the reactivation of viruses which replication is usually kept under control by the immune system. In the case of β-HPV, that is a virus normally present on the skin of individual, its replication is maintained under control by the immune system. When a situation of immunosuppression occurs, the immune system fails to control the replication. Researchers to date suspect that development of SCC is attribute to β-HPV, but yet a link was missing. Previous works proved the presence of β-HPV viral protein and genome amplification in premalignant lesion of human skin. Moreover, a mouse model expressing the early genes of HPV8 (CER-HPV8), spontaneously develop SCC and has been used in the action. We have previously identified that there is an expansion of Lrig1 HF-JZ-KSC population in CER-HPV8 and also in the human field cancerization lesion.

In this project I have taken observations on the aforementioned mouse model of viral induced skin carcinogenesis to unpick the biology of field cancerisation. The mouse models either express all the 5 early region genes from β-HPV (HPV8-CER) or individual early region genes (HPV8-E2, HPV8-E6 HPV8-E6/E7).

With the action, I investigated the molecular mechanism involved in the reactivation of β-HPV and in more detail:
Obj. 1 Identify the HPV8 early region gene that is responsible for ΔNp63 overexpression.
Obj. 2. Determine which signalling pathway(s) is/are activated in HPV8tg-CER mice, resulting in
ΔNp63 overexpression.
Obj. 3. Determine the pathway(s) is/are responsible for ΔNp63 overexpression in human skin FC
Obj. 4. Determine whether immunosuppression promotes SCC formation in HPV8 transgenic mice.
In WP1, I successfully identified HPV8-E6 as the individual HPV8 early region gene responsible for expansion of the novel Lrig1 defined hair follicle junctional zone keratinocyte stem cell (HF-JZ-KSC) population. Moreover, I have found the E6 gene is associated with KSC proliferation through expression of ΔNp63.
In WP2 I have sought to determine which signalling pathway(s) is/are perturbed in HPV8tg-CER mice, resulting in ΔNp63 overexpression. Taking the advantage of a high-throughput screening technique, RNA-Seq, I identified STAT3 as the pathway upregulated in the Lrig1 population of HPV8 mice compared to the control population CD34. In addition, as crucial validation for our findings, I have studied HPV8tg-CER:STAT3+/- mice, that are hemizygous for the STAT3 gene and are less prone to tumorigenesis, enabling assessment of the Lrig1 HF-JZ-KSC. As expected, there was no expansion of the level of Lrig1 population in the CER-HPV8:STAT3+/- mice, and the population was comparable to WT.

In WP3 I planned to translate the finding of WP2 to the human context and determine the signalling pathway(s) responsible for ΔNp63 overexpression in human skin FC. I have selected the samples that show cytopathic effect and detect the presence β-HPV genotypes using an RHA analysis that allow inspect the DNA level and immunofluorescence analysis that observe the protein level. Yet there was not the possibility to accomplish some part of the WP3 such as analyse the pathway involved in the human AK.

Lastly, I completed WP4 in which I determined whether immunosuppression promotes SCC formation in HPV8 transgenic mice. I completed the breeding of HPV8tg-CER with Rag2-KO mice (which lack B and T cells), in order to recreate a mouse model that recapitulate the effect of immunosuppression on HPV8 infection. In the HPV8tg-CER:Rag2KO the size of Lrig1 population increased when compared to the normal counterpart such as Rag2KO only. Furthermore, I compared the rate and frequency of tumour formation in HPV8tg mice versus HPV8tg-CER:Rag2KO deficient mice. Strikingly, a large fraction of HPV8tg-CER:Rag2KO mice developed papilloma at earlier time points when compared to HPV8tgCER mice.
In closing, the work that has been completed during the action, using mouse models of HPV8 (HPV8tg-CER, HPV8tg-E2, HPV8tg-E6, HPV8tg-E7), enable the identification of E6 of β-HPV as the early region gene involved in the expansion of the Lrig1 HF-JZ-KSC population through increased ΔNp63 expression.

The work undertaken has contributed to the state of the art of the keratinocytes stem cell field.

The plan for dissemination and exploitation of results has been fully realized and exceeded.
During the action I have developed a strong network with scientist in the field. This has been possible through the attendance and active participation with work presentation to several conferences.

These conferences have allowed me to meet with the collaborators that kindly offered the mice (Professor Akgul, Professor Gariglio) and the cell line (professor Smola) stated in the action.

I have taken part in many public engagement activities throughout the project, including “FUTURES”, the European Researchers’ Night at the @we the curious event held in Bristol. I also took part in the application for get funds for the first European Researchers’ Night to be held in Cardiff that has recently been awarded.
This action, giving the opportunity to learn new scientific skills and reinforce management and soft skills, it has having a strong impact and potential on my career. I have been deeply immersed in the project throughout these two years: this challenged me to improve as a researcher through managing this work semi-autonomously. I had the privilege to deal with the responsibility to complete the action, and this help me a lot in growing as a scientist and person, enhance my passion for science and my ambitions. Moreover, the precious experience and feedbacks I have got in helping the student gave me confidence and ability in dealing with teaching and project supervision. The collaborations I establish throughout the project has having a massive impact on the scientific part of the action.
Furthermore, in the frame of build my future career and star getting independency, I participate in the writing of two grants as a project group member. One application was successful. The other one is still under revision. I am also in the process of writing the manuscript relative to the research done during the action and aim to submit to a high impact factor journal.
Moreover, the action linked the epithelial carcinoma and HPV8 infection to the stem cell field, providing advancement in the knowledge of a disease like Squamous Cell Carcinoma that has from 60 to 250 fold increase incidence in Organ Transplant Recipience compare to healthy people. Understanding this relationship will have a massive impact on the socio-economical side of the health system: development of a drug that compete with this particular pathway and block the SCC formation.