MECHANISMS OF HUMAN PAPILLOMAVIRUS TYPE 16 E7 (HPV-16 E7)-INDUCED DISRUPTION OF CENTRIOLE DUPLICATION CONTROL

Infection with high-risk human papillomaviruses (HPVs) is the main cause of cervical cancer, the second most common cause of cancer-related mortality in women worldwide. High-risk HPV types, such as HPV-16, express two oncoproteins, E6 and E7, which function to subvert critical host cell cycle control mechanisms in order to promote viral genome amplification. Disruption of the pRB signaling axis and the p53-mediated stress response by the HPV E7 and E6 oncoproteins, respectively, results not only in aberrant proliferation but also in host cellular changes that can promote genomic instability. The high-risk HPV-16 E7 oncoprotein was found to induce centrosome abnormalities thereby disrupting mitotic fidelity and increasing the risk for chromosome missegregation and aneuploidy. Aneuploidy is frequently found in pre-malignant high-risk HPV-associated lesions and is a critical factor for malignant progression. This thesis was designed to determine the molecular mechanisms behind the ability of HPV-16 E7 to rapidly induce centriole overduplication. This rapid induction was found to be possible through the simultaneous formation of more than one daughter centriole at single maternal centrioles (centriole multiplication). It was previously discovered that the centriole multiplication pathway relied on cyclin E, CDK2 and PLK4. However, it was not known before how these molecular players cooperate in the centriole multiplication pathway or how HPV-16 E7 expression promotes the activation of this pathway. Here, we report that cyclin E/CDK2 mediates the aberrant recruitment of PLK4 to maternal centrioles. This initial recruitment step was not sufficient to induce centriole multiplication unless PLK4 protein levels were increased. We found that PLK4 protein levels were controlled by proteolysis, specifically by CUL1-based E3 ubiquitin ligase complexes localized at maternal centrioles. SCF activity was found to control not only baseline PLK4 protein stability but its activity-dependent degradation following cyclin E/CDK2 overexpression. High-risk HPV-16 E7 is known to deregulate cyclin E/CDK2 complexes and we found that ectopic expression of HPV-16 E7 promoted the aberrant recruitment of PLK4 to maternal centrioles. Since our previous experiments have shown that aberrant recruitment of PLK4 is not sufficient to drive centriole overduplication, we determined whether HPV-16 E7 may also disrupt PLK4 expression. We found that HPV-16 E7, but not low-risk HPV proteins or mutants of HPV-16 E7 that lack the ability to induce centriole overduplication, causes a moderate but significant upregulation of PLK4 mRNA. Besides centriole duplication control, we discovered that proteolysis also regulates other aspects of centriole synthesis such as regulation of daughter centriole length. Defining the precise molecular circuitry of centriole biogenesis will aid not only in deepening the current understanding of centriole biogenesis but also aid in identification of novel targets, such as CDK2 or PLK4, for small molecules to prevent centriole abnormalities, mitotic infidelity and malignant progression in pre-invasive high-risk HPV-associated lesions

The HPV-16 E7 oncoprotein induces centriole multiplication through deregulation of Polo-like kinase 4 expression

<p>Abstract</p> <p>Background</p> <p>Infection with high-risk human papillomaviruses (HPVs) such as HPV-16 is intimately associated with squamous cell carcinomas (SCCs) of the anogenital tract and a subset of oropharyngeal carcinomas. Such lesions, including pre-invasive precursors, frequently show multipolar mitoses and aneuploidy. The high-risk HPV-16-encoded E7 oncoprotein has been shown to rapidly induce centrosome abnormalities thereby causing the formation of supernumerary mitotic spindle poles and increasing the risk for chromosome missegregation. HPV-16 E7 has been found to rapidly induce centriole overduplication, in part, through the simultaneous formation of more than one daughter centriole at single maternal centrioles (centriole multiplication). The precise molecular mechanism that underlies HPV-16 E7-induced centriole multiplication, however, remains poorly understood.</p> <p>Findings</p> <p>Here, we show that human keratinocytes engineered to stably express the HPV-16 E7 oncoprotein exhibit aberrant Polo-like kinase 4 (PLK4) protein expression at maternal centrioles. Real-time quantitative reverse transcriptase (qRT-PCR) analysis of these cells revealed an increase of PLK4 mRNA levels compared to control cells. Importantly, the ability of the HPV-16 E7 oncoprotein to induce centriole multiplication was found to correlate with its ability to activate the PLK4 promoter and to up-regulate PLK4 mRNA.</p> <p>Conclusions</p> <p>These results highlight the critical role of PLK4 transcriptional deregulation in centriole multiplication in HPV-16 E7-expressing cells. Our findings encourage further experiments to test transcriptional inhibitors or small molecules targeting PLK4 to prevent centriole abnormalities, mitotic infidelity and malignant progression in HPV-associated neoplasms and other tumors in which PLK4 regulation is disrupted.</p

A novel role of the aryl hydrocarbon receptor (AhR) in centrosome amplification - implications for chemoprevention

<p>Abstract</p> <p>Background</p> <p>Centrosome aberrations can cause genomic instability and correlate with malignant progression in common human malignancies such as breast and prostate cancer. Deregulation of cyclin/cyclin-dependent kinase 2 (CDK2) activity has previously been shown to be critically involved in centrosome overduplication. We therefore test here whether small molecule CDK inhibitors derived from the <it>bis</it>-indole indirubin can be used to suppress centrosome aberrations as a novel approach to chemoprevention of malignant progression.</p> <p>Results</p> <p>As expected, we found that the CDK inhibitor indirubin-3'-oxime (IO) suppresses centrosome amplification in breast cancer cells. However, we made the unexpected discovery that indirubin-derived compounds that have been chemically modified to be inactive as kinase inhibitors such as 1-methyl-indirubin-3'-oxime (MeIO) still significantly reduced centrosome amplification. All indirubins used in the present study are potent agonists of the aryl hydrocarbon receptor (AhR), which is known for its important role in the cellular metabolism of xenobiotics. To corroborate our results, we first show that the coincidence of nuclear AhR overexpression, reflecting a constitutive activation, and numerical centrosome aberrations correlates significantly with malignancy in mammary tissue specimens. Remarkably, a considerable proportion (72.7%) of benign mammary tissue samples scored also positive for nuclear AhR overexpression. We furthermore provide evidence that continued expression of endogenous AhR is critical to promote centriole overduplication induced by cyclin E and that AhR and cyclin E may function in the same pathway. Overexpression of the AhR in the absence of exogenous ligands was found to rapidly disrupt centriole duplication control. Nonetheless, the AhR agonists IO and MeIO were still found to significantly reduce centriole overduplication stimulated by ectopic AhR expression.</p> <p>Conclusions</p> <p>Our results indicate that continued expression of endogenous AhR promotes centrosome amplification in breast cancer cells in a pathway that involves cyclin E. AhR agonists such as indirubins inhibit centrosome amplification even when stimulated by ectopic expression of the AhR suggesting that these compounds are potentially useful for the chemoprevention of centrosome-mediated cell division errors and malignant progression in neoplasms in which the AhR is overexpressed. Future studies are warranted to determine whether individuals in which nuclear AhR overexpression is detected in benign mammary tissue are at a higher risk for developing pre-cancerous or cancerous breast lesions.</p

Spatial niche formation but not malignant progression is a driving force for intratumoural heterogeneity

Intratumoural heterogeneity (ITH) is a major cause of cancer-associated lethality. Extensive genomic ITH has previously been reported in clear cell renal cell carcinoma (ccRCC). Here we address the question whether ITH increases with malignant progression and can hence be exploited as a prognostic marker. Unexpectedly, precision quantitative image analysis reveals that the degree of functional ITH is virtually identical between primary ccRCCs of the lowest stage and advanced, metastatic tumours. Functional ITH was found to show a stage-independent topological pattern with peak proliferative and signalling activities almost exclusively in the tumour periphery. Exome sequencing of matching peripheral and central primary tumour specimens reveals various region-specific mutations. However, these mutations cannot directly explain the zonal pattern suggesting a role of microenvironmental factors in shaping functional ITH. In conclusion, our results indicate that ITH is an early and general characteristic of malignant growth rather than a consequence of malignant progression