Human Papillomavirus (HPV) is a small, double stranded DNA tumour virus. Infection with HPV normally results in formation of warts. Certain types of HPV, such as type -16 and -18, are shown to have a causal role in the development of uterine cervical cancer, and are so called high risk type HPV. Recently, a role of HPV in breast cancer has been suggested, although a causal role for HPVs in human breast cancer is yet to be demonstrated. The first part of this study investigates the association of HPV with human breast cancer. The results demonstrate that 48% of breast cancers that occurred in Australian women are HPV positive and they are mainly variants of HPV-18. Further analysis shows that HPV positive breast cancer patients are significantly younger than HPV negative patients, suggesting infection with HPV increases the risk of breast cancer development. This is coincidental with increased risk of HPV infection in sexually active young women and provides evidence that HPV has a role in breast cancer development. The second part of this project investigates the mechanisms by which high risk type HPV oncogenic protein E6, transforms primary human foreskin keratinocytes (natural host cells of HPV). HPV E6 is always expressed in HPV positive cervical carcinoma and results in the degradation of the cellular tumour suppressor protein p53. It is generally believed that HPV E6 contributes to HPV transformation by degradation of p53 protein which leads to cellular immortalisation ? an early step in tumorigenic transformation. Subsequent studies, however, indicate that HPV E6 possesses other functions (such as induction of telomerase activity) which may also be involved in cellular immortalisation. The results of my investigations demonstrate: 1) that degradation of p53 protein is required but is insufficient to immortalise primary cells; 2) that HPV E6 induced telomerase activity is coincidental with an increase in cell culture passage number; 3) that multiple functions of high risk type HPV E6 protein are required for cellular immortalisation. This finding suggests HPV infection is associated with early onset of breast cancer and that multiple functions of high risk type HPV E6 protein are involved in cellular immortalisation. Further study in both of these areas should provide alternative diagnostic markers, leading to prevention and treatment strategies for HPV positive breast cancer and other cancers.

Human Papillomavirus (HPV) is a small, double stranded DNA tumour virus. Infection with HPV normally results in formation of warts. Certain types of HPV, such as type -16 and -18, are shown to have a causal role in the development of uterine cervical cancer, and are so called high risk type HPV. Recently, a role of HPV in breast cancer has been suggested, although a causal role for HPVs in human breast cancer is yet to be demonstrated. The first part of this study investigates the association of HPV with human breast cancer. The results demonstrate that 48% of breast cancers that occurred in Australian women are HPV positive and they are mainly variants of HPV-18. Further analysis shows that HPV positive breast cancer patients are significantly younger than HPV negative patients, suggesting infection with HPV increases the risk of breast cancer development. This is coincidental with increased risk of HPV infection in sexually active young women and provides evidence that HPV has a role in breast cancer development. The second part of this project investigates the mechanisms by which high risk type HPV oncogenic protein E6, transforms primary human foreskin keratinocytes (natural host cells of HPV). HPV E6 is always expressed in HPV positive cervical carcinoma and results in the degradation of the cellular tumour suppressor protein p53. It is generally believed that HPV E6 contributes to HPV transformation by degradation of p53 protein which leads to cellular immortalisation ? an early step in tumorigenic transformation. Subsequent studies, however, indicate that HPV E6 possesses other functions (such as induction of telomerase activity) which may also be involved in cellular immortalisation. The results of my investigations demonstrate: 1) that degradation of p53 protein is required but is insufficient to immortalise primary cells; 2) that HPV E6 induced telomerase activity is coincidental with an increase in cell culture passage number; 3) that multiple functions of high risk type HPV E6 protein are required for cellular immortalisation. This finding suggests HPV infection is associated with early onset of breast cancer and that multiple functions of high risk type HPV E6 protein are involved in cellular immortalisation. Further study in both of these areas should provide alternative diagnostic markers, leading to prevention and treatment strategies for HPV positive breast cancer and other cancers.

Cellular immortality is one of the ten hallmarks of human cancer and has been shown to be an essential prerequisite for malignant progression (Hanahan and Weinberg., 2011, Newbold et al., 1982, Newbold and Overell., 1983). In contrast, normal human somatic cells proliferate for a limited number of population doublings before entering permanent growth arrest known as replicative senescence. This is thought to be due to the progressive shortening of telomeric sequences with each round of cell division. Over 90% of human tumours, but not the majority of human somatic cells, have been found to express telomerase activity (Kim et al., 1994). The rate-limiting component of the human telomerase enzyme is the telomerase reverse transcriptase subunit, which is encoded by the hTERT gene. Transfection of hTERT cDNA into normal human fibroblasts and epithelial cells may sometimes be sufficient to confer cellular immortality (Newbold., 2005, Stampfer and Yaswen., 2002). Therefore, de-repression of hTERT and telomerase re-activation are thought to be critical events in human carcinogenesis and is the predominant mechanism by which cancer cells maintain their proliferative capacity. Previously, our group has shown that introduction of a normal, intact copy of human chromosome 3 into the 21NT primary breast cancer cell line by microcell-mediated monochromosome transfer (MMCT), is associated with strong telomerase repression and induction of cell growth arrest within the majority of hybrid clones (Cuthbert et al., 1999). Structural mapping of chromosome 3 within telomerase-positive revertent clones revealed two regions of deletion: 3p21.3-p22 and 3p12-p21.1, thought to harbour the putative telomerase repressor sequence(s). Subsequent studies showed that the chromosome 3p-encoded telomerase repressor sequence(s) mediates its function by means of transcriptional silencing of hTERT, in part, through chromatin remodelling of two sites within intron 2 of the hTERT gene (Ducrest et al., 2001, Szutorisz et al., 2003). Attempts to achieve positional cloning of hTERT repressor sequences on chromosome 3p identified two interesting candidates; the histone methyltransferase SETD2 and an adjacent long non-coding RNA (lncRNA) sequence known as FLJ/KIF9-AS1 (Dr. T. Roberts, unpublished data). Through MMCT-mediated introduction of intact chromosomes 3 and 17 into the 21NT cell line, I have demonstrated that at least two as yet unidentified telomerase repressor sequences (one located on each of these two normal chromosomes) may function to repress telomerase activity within the same breast cancer cell line, which suggests that multiple, independent telomerase regulatory pathways may be inactivated within the same cancer type. Furthermore, by examining the consequences of forced SETD2 and FLJ expression within the 21NT cell line, together with siRNA-mediated knockdown of SETD2 within a single telomerase-repressed 21NT-chromosome 3 hybrid, I have provided evidence to show that neither of these two candidate genes may function as a regulator of hTERT transcription. Through interrogation of relevant literature, a set of four candidate 3 telomerase regulatory genes (BAP1, RASSF1A, PBRM1 and PARP-3) were selected for further investigation based on their location within the 3p21.1-p21.3 region together with their documented role in the epigenetic regulation of target gene expression. Using mammalian expression vectors containing candidate gene cDNA sequences, my colleague Dr. T. Roberts and I demonstrated that forced overexpression of BAP1 and PARP-3 within the 21NT cell line is associated with consistent, but not always sustained, repression of hTERT transcriptional activity and telomerase activity. It is therefore possible that at least two sequences may exist on chromosome 3p that function collectively to regulate hTERT expression within breast cancer cells. Finally, using an in vitro model of human mammary epithelial cell (HMEC) immortalization, involving the targeted abrogation of two pathologically relevant genes, p16 and p53 to generate a series of variant clones at different stages of immortal transformation (developed by my colleague Dr. H. Yasaei), I have shown that single copy deletions on chromosome 3p are a frequent, clonal event, specifically associated with hTERT de-repression and immortal transformation. Subsequent high-density single nucleotide polymorphism (SNP) array analysis of immortal variants carried out by Dr. H. Yasaei, identified a minimal common region of deletion localized to 3p14.2-p22. Together, these findings provide additional evidence to show that chromosome 3p may harbour critical hTERT repressor sequences, that are lost as an early event during breast carcinogenesis.