Noel J. Whitaker

Human papillomavirus-induced carcinogenesis and the ubiquitin–proteasome system

Certain types of human papillomaviruses have been etiologically associated with malignant lesions, most notably with cervical cancer. The major oncoproteins of these cancer-associated viruses are encoded by the viral E6 and E7 genes. Thorough characterization of these oncoproteins and their interaction with cellular proteins has shown that both E6 and E7 exploit the ubiquitin-proteasome system to degrade and, thus, to functionally inactivate negative cell-regulatory proteins including members of the p110(RB) family and p53. This act of piracy is assumed to contribute to both the efficient propagation of HPVs and HPV-induced carcinogenesis.

Human papilloma virus is associated with breast cancer

Background:There is increasing evidence that high-risk human papilloma virus (HPV) is involved in cancers in addition to cervical cancer. For example, it is generally accepted that HPV has a role in a significant proportion of head and neck tumours, and it has long been hypothesised that hormone dependent oncogenic viruses, such as HPV may have causal roles in some human breast cancers. A number of reports have identified HPV DNA in breast tissue and breast cancer specimens, but these rely on standard polymerase chain reaction (PCR), which is criticised for its propensity for contamination.Methods:We have used two different technologies, in situ and standard PCR (with sequencing), and histology based on light microscopy.Results:We unambiguously demonstrate the presence of high-risk HPV in the cells of breast cancer specimens and breast cancer cell lines. In addition, we also show that the oncogenic characteristics of HPV associated breast cancer are very similar to HPV-associated cervical cancer. Specifically, that putative koilocytes are present in some HPV associated breast cancers.Interpretation:The above observations indicate a likely causal role for high-risk HPV in human breast cancer and offer the possibility of primary prevention of some breast cancers by vaccination against HPV.

Identification of human papillomavirus DNA gene sequences in human breast cancer

Human papilloma viruses (HPVs) are accepted as being carcinogenic in human cervical and anogenital cancers. The suspicion that HPVs may also have a role in human breast cancer is based on the identification of HPVs in human breast tumours and the immortalisation of normal human breast cells by HPV types 16 and 18. For this investigation, DNA that had been previously extracted and fresh frozen at −70°C from 50 unselected invasive ductal breast cancer specimens were screened by polymerase chain reaction (PCR) for HPV type 16, 18 and 33 gene sequences. We show that HPV 18 gene sequences are present in DNA extracted from breast tumours in Australian women. Overall, 24 (48%) of the 50 samples were HPV positive. Overall no correlations with tumour grade, patient survival, steroid receptor status, ERB-2, p53 expression and mutation were observed. Human papilloma viruses may have a role in human breast cancer. We speculate that HPVs may be transmitted by hand from the female perineum to the breast.

Epstein-Barr Virus, Human Papillomavirus and Mouse Mammary Tumour Virus as Multiple Viruses in Breast Cancer

Background The purpose of this investigation is to determine if Epstein Barr virus (EBV), high risk human papillomavirus (HPV), and mouse mammary tumour viruses (MMTV) co-exist in some breast cancers. Materials and Methods All the specimens were from women residing in Australia. For investigations based on standard PCR, we used fresh frozen DNA extracts from 50 unselected invasive breast cancers. For normal breast specimens, we used DNA extracts from epithelial cells from milk donated by 40 lactating women. For investigations based on in situ PCR we used 27 unselected archival formalin fixed breast cancer specimens and 18 unselected archival formalin fixed normal breast specimens from women who had breast reduction surgery. Thirteen of these fixed breast cancer specimens were ductal carcinoma in situ (dcis) and 14 were predominantly invasive ductal carcinomas (idc). Results EBV sequences were identified in 68%, high risk HPV sequences in 50%, and MMTV sequences in 78% of DNA extracted from 50 invasive breast cancer specimens. These same viruses were identified in selected normal and breast cancer specimens by in situ PCR. Sequences from more than one viral type were identified in 72% of the same breast cancer specimens. Normal controls showed these viruses were also present in epithelial cells in human milk – EBV (35%), HPV, 20%) and MMTV (32%) of 40 milk samples from normal lactating women, with multiple viruses being identified in 13% of the same milk samples. Conclusions We conclude that (i) EBV, HPV and MMTV gene sequences are present and co-exist in many human breast cancers, (ii) the presence of these viruses in breast cancer is associated with young age of diagnosis and possibly an increased grade of breast cancer.

Induction of the p53-target gene GADD45 in HPV-positive cancer cells.

The E6 oncoprotein of human papillomaviruses (HPVs) has the potential to functionally antagonize p53. In several experimental model systems, ectopic expression of E6 can block the genotoxic induction of the growth inhibitory p53 target gene gadd45, suggesting that the inactivation of this pathway may play a major role for HPV-associated cell transformation. Here, we investigated whether this reflects the regulation of gadd45 expression in carcinoma-derived HPV-positive cells. We found that the gadd45 gene is efficiently induced by mitomycin C, cisplatin, and UV irradiation in a series of HPV-positive cervical cancer cell lines. Moreover, clear induction of gadd45 gene expression was also observed following treatment with γ-irradiation, a pathway that is strictly dependent on functional p53. This contrasted with findings in human foreskin keratinocytes experimentally immortalized by expressing the HPV16 E6, E7, or E6/E7 oncogenes from the heterologous CMV promoter, where expression of the E6 gene was linked to a lack of gadd45 induction following γ-irradiation. These results indicate (1) that the tumorigenic phenotype of HPV-positive cancer cells is not linked to an inability to induce the gadd45 gene following DNA damage, (2) that experimental model systems in which the E6 gene is expressed ectopically and/or in a different cellular context do not necessarily reflect the regulation of p53-associated pathways in HPV-positive cancer cells and (3) that a pathway strictly depending on functional p53 is inducible in HPV-positive cancer cells, providing direct evidence that the endogenous p53 protein in these cells is competent to activate a cellular target gene, despite coexpression of the viral E6 oncogene.

Viruses and human breast cancer

There are well-established risk factors for breast cancer, most of which relate to estrogens and growth hormones in females. These include early-age menarche, late-age menopause, postmenopausal obesity and use of hormone therapy. However, these factors do not account for the sixfold difference in breast cancer incidence and mortality between countries and the fact that these differences dramatically lessen after migration; nor do they account for male breast cancer. Accordingly, hormone-responsive viruses have become major suspects as etiological agents for human breast cancer. Human papillomaviruses, mouse mammary tumor virus and Epstein-Barr virus are the prime candidate viruses as causes of human breast cancer. Human papillomaviruses and the mouse mammary tumor virus have hormone responsive elements that appear to be associated with enhanced replication of these viruses in the presence of corticosteroid and other hormones. This biological phenomenon is particularly relevant because of the hormone dependence of breast cancer. Viral genetic material for each of these candidate viruses has been identified by polymerase chain reaction in breast tumors but rarely in normal breast tissue controls. Pooled data from controlled studies show substantial odds ratios for the presence of viral genetic material in breast tumors compared with normal controls. These and additional data provide substantial, but not conclusive, evidence that human papillomavirus, the mouse mammary tumor virus and Epstein-Barr virus may have a role in the etiology of human breast cancer. If conclusive evidence for a role of these viruses in breast carcinogenesis can be developed, there is a practical possibility of primary prevention.

Cytotoxic effects of antipsychotic drugs implicate cholesterol homeostasis as a novel chemotherapeutic target

The reported reduction in cancer risk in those suffering from schizophrenia may be because antipsychotic medications have antineoplastic effects. In this study, 6 antipsychotic agents with a range of structural and pharmacological properties (reserpine, chlorpromazine, haloperidol, pimozide, risperidone and olanzapine), were screened for their effect on the viability of cell lines derived from lymphoblastoma, neuroblastoma, non‐small cell lung cancer and breast adenocarcinoma. We aimed to determine if antipsychotic drugs in general possess cancer‐specific cytotoxic potential, and whether it can be attributed to a common mode of action. With the exception of risperidone, all drugs tested displayed selective inhibition of the viability of cancer cell lines compared with normal cells. Using Affymetrix expression microarrays and quantitative real‐time polymerase chain reaction, we found that for the antipsychotic drugs, olanzapine and pimozide, cytotoxicity appeared to be mediated via effects on cholesterol homeostasis. The role of cholesterol metabolism in the selective cytotoxicity of these drugs was supported by demonstration of their increased lethality when coadministered with a cholesterol synthesis inhibitor, mevastatin. Also, pimozide and olanzapine showed accelerating cytotoxic effects from 12 to 48 hr in time course studies, mirroring the time‐dependent onset of cytotoxicity induced by the amphiphile, U18666A. On the basis of these results, we concluded that the Class II cationic amphiphilic properties of antipsychotic drugs contribute to their cytotoxic effects by acting on cholesterol homeostasis and altering the biophysical properties of cellular membranes, and that drugs affecting membrane‐related cholesterol pathways warrant further investigation as potential augmentors of standard cancer chemotherapy.

Koilocytes indicate a role for human papilloma virus in breast cancer

Background:High-risk human papilloma viruses (HPVs) are candidates as causal viruses in breast cancer. The scientific challenge is to determine whether HPVs are causal and not merely passengers or parasites. Studies of HPV-related koilocytes in breast cancer offer an opportunity to address this crucial issue. Koilocytes are epithelial cells characterised by perinuclear haloes surrounding condensed nuclei and are commonly present in cervical intraepithelial neoplasia. Koilocytosis is accepted as pathognomonic (characteristic of a particular disease) of HPV infection. The aim of this investigation is to determine whether putative koilocytes in normal and malignant breast tissues are because of HPV infection.Methods:Archival formalin-fixed normal and malignant breast specimens were investigated by histology, in situ PCR with confirmation of the findings by standard PCR and sequencing of the products, plus immunohistochemistry to identify HPV E6 oncoproteins.Results:human papilloma virus-associated koilocytes were present in normal breast skin and lobules and in the breast skin and cancer tissue of patients with ductal carcinoma in situ (DCIS) and invasive ductal carcinomas (IDCs).Interpretation:As koilocytes are known to be the precursors of some HPV-associated cervical cancer, it follows that HPVs may be causally associated with breast cancer.

Restoration of p53 expression sensitizes human papillomavirus type 16 immortalized human keratinocytes to CD95-mediated apoptosis

To understand the function of the individual oncogenes of HPV16 in modulating the cellular response to apoptogenic signals, we used human keratinocytes immortalized with either E6, E7 or E6/E7 oncoproteins as model system. Applying CD95 antibodies or recombinant CD95 ligand, only the E7-immortalized cells underwent extensive apoptosis. In contrast, E6- and E6/E7-expressing keratinocytes were resistant. Dominance of E6 correlated with significant down-regulation of p53, c-Myc, p21 and Bcl-2. CD95 was found to be reduced in resistant HPV-positive cells, while there were no quantitative differences in expression levels of FADD, FLICE/caspase-8 or caspase-3. Notably, in contrast to primary human keratinocytes, all immortalized cells showed a general reduction of c-FLIP, an inhibitory protein which normally prevents unscheduled CD95-induced apoptosis. E6- and E6/E7-positive keratinocytes, however, can be sensitized to CD95 apoptosis by blocking proteasome-mediated proteolysis. CD95-resistant HPV-positive cells underwent apoptosis within 3–5 h upon co-incubation with MG132 and agonistic antibodies or CD95 ligand, which was preceded by a strong re-expression of p53 and c-Myc, but not of other half-life controlled proteins such as Bax or IκBα. Blockage of proteasomal activity alone did not result in apoptosis, although the same set of pro-apoptotic proteins was up-regulated. Performing similar experiments with cervical carcinoma cells expressing mutated p53 (C33a) or with p53-‘null’ lung carcinoma cells (H1299), no CD95 cell killing occurred eventhough c-Myc was strong induced. These data indicate that the reduced bioavailability of p53 is a key-regulatory event in perturbation of CD95 signaling in HPV16 immortalized keratinocytes.

Mouse Mammary Tumor Virus–like Sequences in Human Breast Cancer

Mouse mammary tumor virus (MMTV) sequences have been reported to be present in some human breast cancers, but it is unclear whether they have any causal role. In mice, MMTV promotes tumor formation indirectly by insertional mutagenesis of Wnt oncogenes that lead to their activation. In this study, we investigated the status of Wnt-1 in human breast cancers harboring MMTV-like sequences encoding viral envelope (env) genes. We confirmed the detection of env sequences in the nucleus of human breast cancer specimens that are similar in appearance to mouse mammary tumors expressing MMTV env sequences. MMTV env sequences in human breast cancers were also nearly indistinguishable from env sequences in mouse MMTV isolates. Further, Wnt-1 expression was higher in specimens of env-positive ductal carcinoma in situ and invasive ductal carcinoma, relative to env-negative specimens. Our findings extend the evidence that MMTV sequences found in naturally occurring mouse mammary tumors can be found in some human breast cancers, prompting further evaluation of causal roles in these settings.