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Research Statement

Introduction:

My laboratory is interested in the differentiation of human keratinocytes (skin cells) and determining the important pathways required for controlled stratification and differentiation. We have used Human Papillomavirus type 16 (HPV-16) to delineate pathways that are important for differentiation, as these viruses only infect epithelial cells and inhibit their differentiation. Using a novel in vitro 3-dimensional culture system that recapitulates the morphological and biochemical changes observed in skin, we have been able to study the differentiation process in a physiologically relevant manner.

Keratinocyte differentiation can be inhibited using two viral proteins from HPV-16, called E6 and E7. A consequence of the inhibition of differentiation is that the cells become immortalized. Another consequence is that the cells exhibit genomic instability, with the production of tetraploidy and eventually aneuploidy. We investigated the function of E6 and E7 that were required for immortalization, as this would indicate important pathways for normal differentiation. E6 and E7 have a number of important functions. We had previously shown that the ability of E7 to abrogate Rb function was important for immortalization and inhibition of keratinocyte differentiation. E6 is best known for the ability to bind and degrade p53. However, it can cause the degradation of other proteins, such as the PDZ containing proteins the membrane-associated guanylate kinase homologues (MAGUK), which are found at the membrane and are thought to be involved in intra- and inter-cellular interactions and cell polarization. It can also transcriptionally regulate proteins such as the telomerase.

The following is a short description of our research over the last 3 years which builds on the observations we have made using the HPV-16 E6 and E7 proteins.

Retinoblastoma protein (pRb) and keratinocyte differentiation:

pRb, the prototypical member, is involved in cell cycle regulation and is mutated in the majority of carcinomas. However, increasing evidence suggests that pRb, but not the other two family members, p107 and p130, is also important for the differentiation of some cell types, including human keratinocytes. In the light of these results, we are now investigating the role of the pRb in keratinocyte differentiation at the molecular level. We have found that pRb, binds to a cellular histone acetyltransferase, called P/CAF, in in vivo and in vitro experiments. P/CAF can acetylate Rb in the C-terminal domain on amino acids 873 and 874. In vivo studies have shown that both P/CAF and the p300/CBP acetyl transferases can synergize to acetylate pRb. Using mouse muscle cells which are null for pRb we have shown that pRb acetylation occurs during differentiation and is not present in cycling cells. Acetylation of pRb is required for the activation of MyoD, a master transcription factor, necessary for the activation of downstream muscle specific genes. Acetylation of pRb is also required for permanent cell cycle arrest and for downstream stimulation of muscle specific differentiation factors. Similar experiments have been carried out in human keratinocytes, using RNAi to reduce levels of pRb, since null cells are not available. In the situation of reduced Rb, human keratinocytes fail to exit the cell cycle permanently and there is an inhibition of differentiation as observed by the lack of differentiation-specific markers. The ability of pRb to arrest both muscle and keratinocyte cells relies on the ability to bind the E3 ligase mdm2 and EID-1 (E1A inhibitor of differentiation). The later protein when over-expressed causes inhibition of differentiation of mouse muscle cells.

p63 in cancer and keratinocyte differentiation:

p63 belongs to the same family of proteins as p53 and p73 and like these other family members, it has several isoforms. Like the other family members, p63 can transcriptionally activate genes and the genes activated by p63 are similar to those that respond to p53. The p63 isoforms are required for development and differentiation of a number of organs including skin and it is unregulated in carcinomas. While investigating functions of E6 we discovered that the levels of p63 in the epithelium varied depending on the ability of E6 to degrade p53. The function of each of the 6 isoforms of p63 is unknown and E6 expression increases the levels of 3 of the 6 isoforms. We have knocked down the most common isoform,
DNp63a using RNA interference and find that this inhibits keratinocytes differentiation. We can rescue this phenotype by re-expressing DNp63a and are investigating transcriptional targets this isoform. Because there appears to be an interaction between p53 and p63, we are also keen to determine if the gain of function observed with some naturally occurring p53 mutations in cancer, are due to p63 isoforms.

Studies on the G2/M de-regulation and genomic instability (In collaboration with Dr Daksha Patel):

When HPV-16 E6 and E7 are expressed in primary human keratinocytes, the cells become tetraploidy and after several doublings become aneuploidy. To determine the cause of the genomic instability we used Microarray analysis on primary human keratinocytes expressing E6 and E7and discovered several genes involved in the G2 to M transition and in M-phase that were up-regulated in E6/E7-expressing keratinocytes, compared to normal cells. This is potentially very interesting since evidence suggests that tetraploidy leads to aneuploidy, a result of genomic instability. The polyploidy phenotype is dependent on the ability of E6 to degrade p53 and for E7 to abrogate pRb functions. Many of the genes up-regulated by E6 and E7, such as polo-like kinase 1, Aurora A and B kinases and cdk1 have already been shown to cause mitotic abnormalities when over expressed. One particularly interesting result is that tetraploidy is dependent on the up-regulation of UBCH10, an E2 conjugating enzyme, by E6 and E7. UBCH10 is required for the function of the Anaphase Promoting Complex (APC).

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  • 10 Similar Profiles
Keratinocytes Medicine & Life Sciences
Human papillomavirus 16 Medicine & Life Sciences
Foreskin Medicine & Life Sciences
keratinocytes Agriculture & Biology
Papillomaviridae Agriculture & Biology
DNA Damage Medicine & Life Sciences
Up-Regulation Medicine & Life Sciences
Neoplasms Medicine & Life Sciences

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Projects 2007 2015

R1012CNR: MRC Skills Gap - G900870 - Prof Haigh

McCance, D.

01/08/200813/12/2019

Project: Research

R1310CNR: The role of p63 isoforms in head and neck cancers

McCance, D., James, J. & Patel, D.

01/08/201031/07/2015

Project: Research

Research Output 2002 2018

3 Citations (Scopus)

ΔNp63γ/SRC/Slug signalling axis promotes epithelial-to-mesenchymal transition in squamous cancers

Srivastava, K., Pickard, A., Craig, S., Quinn, G., Lambe, S., James, J., McCance, D. & McDade, S., 08 May 2018, In : Clinical Cancer Research. 24, 16, p. 3917-3927 11 p.

Research output: Contribution to journalArticle

Open Access
File
Gastropoda
Epithelial-Mesenchymal Transition
Neoplasms
Papillomaviridae
Foreskin
10 Citations (Scopus)

p63 drives invasion in keratinocytes expressing HPV16 E6/E7 genes through regulation of Src-FAK signalling

Srivastava, K., Pickard, A., McDade, S. & McCance, D. J., 2017, In : Oncotarget. 8, 10, p. 16202-16219 18 p.

Research output: Contribution to journalArticle

Open Access
File
Matrix Metalloproteinase 14
Focal Adhesion Protein-Tyrosine Kinases
Keratinocytes
Foreskin
Genes
50 Citations (Scopus)

HPV-Related Oropharynx Cancer in the United Kingdom: An Evolution in the Understanding of Disease Etiology

Schache, A. G., Powell, N. G., Cuschieri, K. S., Robinson, M., Leary, S., Mehanna, H., Rapozo, D., Long, A., Cubie, H., Junor, E., Monaghan, H., Harrington, K. J., Nutting, C. M., Schick, U., Lau, A. S., Upile, N., Sheard, J., Brougham, K., West, C. M. L., Oguejiofor, K. & 12 othersThomas, S., Ness, A. R., Pring, M., Thomas, G. J., King, E. V., McCance, D. J., James, J. A., Moran, M., Sloan, P., Shaw, R. J., Evans, M. & Jones, T. M., 15 Nov 2016, In : Cancer Research. 76, 22, p. 6598-6606 9 p.

Research output: Contribution to journalArticle

Oropharyngeal Neoplasms
Squamous Cell Carcinoma
Confidence Intervals
Incidence
United Kingdom
10 Citations (Scopus)

HPV16 Down-Regulates the Insulin-Like Growth Factor Binding Protein 2 to Promote Epithelial Invasion in Organotypic Cultures

Pickard, A., McDade, S. S., McFarland, M., McCluggage, W. G., Wheeler, C. M. & McCance, D. J., Jun 2015, In : PLoS Pathogens. 11, 6, 23 p., 1004988.

Research output: Contribution to journalArticle

Open Access
File
17 Citations (Scopus)

IGF-Binding Protein 2 - Oncogene or Tumor Suppressor?

Pickard, A. & McCance, D. J., Feb 2015, In : Frontiers in endocrinology. 6, 25, p. 25

Research output: Contribution to journalArticle

Open Access
File
Insulin-Like Growth Factor Binding Protein 2
Oncogenes
Neoplasms

Activities 1985 2004

  • 7 Membership of peer review panel or committee
  • 2 Membership of external research organisation

American Cancer Society (External organisation)

Dennis McCance (Member)
20042006

Activity: Membership typesMembership of external research organisation

University of Birmingham (External organisation)

Dennis McCance (Reviewer)
2003 → …

Activity: Membership typesMembership of peer review panel or committee

Indiana University (External organisation)

Dennis McCance (Member)
19992006

Activity: Membership typesMembership of peer review panel or committee

US Department of Defence (External organisation)

Dennis McCance (Reviewer)
19981999

Activity: Membership typesMembership of peer review panel or committee

American Cancer Society (External organisation)

Dennis McCance (Reviewer)
1995 → …

Activity: Membership typesMembership of peer review panel or committee