THE HUMAN PAPILLOMAVIRUS TYPE 16 E7 (HPV-16 E7) ONCOPROTEIN AND THE HOST CELL DNA DAMAGE RESPONSE

High-risk human papillomaviruses (HPVs), such as HPV-16, are the etiological agents of squamous cell carcinomas (SCCs) of the anogenital tract and a subset of oropharyngeal cancers. High-risk HPVs encode two oncoproteins, E6 and E7, which promote unscheduled host cell proliferation by targeting the p53 and pRB tumor suppressor proteins, respectively. HPV-16 E7 has been shown to stimulate structural chromosomal instability and DNA breakage. These findings raise several important questions. First, how does HPV-16 E7 induce DNA damage? Second, what are the precise consequences of HPV-16 E7-induced DNA damage for host cell genomic integrity, and lastly, how do HPV-16 E7-expressing cells maintain proliferation despite activated DNA damage checkpoints? Here, we show that HPV-16 E7 activates the Fanconi Anemia (FA) pathway, a branch of the host cell DNA damage response that primarily responds to stalled DNA replication forks. Importantly, we show that HPV-16 E7 expression in FA-deficient cells accelerates the formation of structural chromosomal alterations, which may help to explain the heightened susceptibility of FA patients to HPV-associated tumors. However, we also provide evidence that HPV-16 E7-induced FA pathway activation in FA-proficient cells may contribute to evasion of anti-proliferative host cell barriers by promoting alternative lengthening of telomeres (ALT). Finally, we demonstrate that HPV-16 E7 circumvents DNA damage checkpoint control and promotes aberrant mitotic entry by increasing the proteolytic turnover of claspin, which plays a role in the ATR/CHK1-mediated replication stress response. Collectively, our results underscore that HPV-16 E7 interferes with host cell genome integrity by inducing DNA replication stress. The detrimental effects of HPV-16 E7 on the genomic integrity of host cells with a deficient FA pathway support the notion that this DNA damage response pathway is crucial to prevent HPV-16 E7-induced genomic instability and malignant progression. However, we also provide evidence that HPV-16 E7 can exploit the FA pathway to promote cellular immortalization. Future experiments to explore these events for cancer therapy and/or prevention are warranted

Investigating the effects of network structure and afferent feedback in models of rhythmic movement

Animals can generate distinct rhythmic behaviors using a shared set of muscles and motoneurons, and in certain cases, the structure responsible for generating these movements is unknown. Distinct networks could be dedicated solely to particular behaviors, a singular network could control various movements through reorganization or under different inputs, or a hybrid of these two concepts could exist. In the first chapter of this thesis, we explore the compatibility of different network characteristics with experimental results regarding swimming and scratching rhythm generation in the turtle. We propose three distinct architectures that represent a range of connectivity between networks responsible for these rhythms, and test their performance against a set of experimental benchmarks regarding dual stimulation. The results of our modeling concur with experimental results, suggesting that networks that generate locomotion and scratching share important components. In the second and third chapters, we focus our attention on a previously published neuromechanical locomotor model. In this closed loop system, a central pattern generator (CPG) establishes a rhythm under sufficient supra-spinal drive and controls the activity of a pendular limb, which sends afferent signals back to the CPG, affecting its operation. Increasing the drive to the CPG increases the limb frequency through changes in the stance phase duration only, which is a key feature of normal overground locomotion. Using geometric singular perturbation theory, we analyze the mechanisms responsible for rhythm generation in the CPG, both in the presence and absence of feedback. We exploit our observations to construct a reduced model that is qualitatively similar to the original, but tractable for rigorous discussion. We prove the existence of a locomotor cycle in this reduced system using a novel version of the Melnikov function, adapted for discontinuous systems. We highlight how the limb dynamics shape overall model behavior, and indicate a crucial relationship between components that controls the model's asymmetric response to drive changes. Finally, we utilize our understanding of the model dynamics to explain its performance under various modifications, including recovery of oscillatory behavior after spinal cord injury and response to changes in load

A dynamical systems analysis of afferent control in a neuro--mechanical model of locomotion

Existing models have represented a locomotor system as a rhythmic driver, or central pattern generator (CPG), coupled to a mechanical limb, with feedback closing the loop. Our collaborators have developed a version of this model in which the CPG establishes a rhythm when activating drive is present, and feedback from ground strike helps control phase switching and rhythm stabilization. Spinal cord injury can be simulated through termination of drive, which ceases the rhythm. We derive and analyze a reduced representation of this model, which will elucidate general principles of phase and frequency control in normal locomotion. We aim to understand how the presence of feedback provides stable locomotion, allowing oscillations at a wider range of drive values to the CPG (specifically at lower values) than the CPG without afferent feedback. Furthermore, increasing the drive intensity to the CPG increases locomotor speed by reducing only the duration of the stance phase, at a relatively constant duration of swing phase [1]. This phase asymmetry has been seen in normal locomotion in cats, rats, and humans, but is not observed in the locomotor model without feedback, so we analyze how feedback facilitates this asymmetry. We will also reveal sufficient conditions for recovering rhythmicity in the injured state - how in the absence of drive, increased feedback can restore locomotion and contribute to locomotor stability, with an analysis of differences in rhythm robustness before and after loss of drive

PIK3CA mutant tumors depend on oxoglutarate dehydrogenase

Oncogenic PIK3CA mutations are found in a significant fraction of human cancers, but therapeutic inhibition of PI3K has only shown limited success in clinical trials. To understand how mutant PIK3CA contributes to cancer cell proliferation, we used genome scale loss-of-function screening in a large number of genomically annotated cancer cell lines. As expected, we found that PIK3CA mutant cancer cells require PIK3CA but also require the expression of the TCA cycle enzyme 2-oxoglutarate dehydrogenase (OGDH). To understand the relationship between oncogenic PIK3CA and OGDH function, we interrogated metabolic requirements and found an increased reliance on glucose metabolism to sustain PIK3CA mutant cell proliferation. Functional metabolic studies revealed that OGDH suppression increased levels of the metabolite 2-oxoglutarate (2OG). We found that this increase in 2OG levels, either by OGDH suppression or exogenous 2OG treatment, resulted in aspartate depletion that was specifically manifested as auxotrophy within PIK3CA mutant cells. Reduced levels of aspartate deregulated the malate-aspartate shuttle, which is important for cytoplasmic NAD + regeneration that sustains rapid glucose breakdown through glycolysis. Consequently, because PIK3CA mutant cells exhibit a profound reliance on glucose metabolism, malate-aspartate shuttle deregulation leads to a specific proliferative block due to the inability to maintain NAD + /NADH homeostasis. Together these observations define a precise metabolic vulnerability imposed by a recurrently mutated oncogene. Keyword: PIK3CA; 2OG; OGDH; TCA cycle; glycolysisDamon Runyon Cancer Research Foundation (HHMI Fellowship

The evolving definition of carcinogenic human papillomavirus

Thirteen human papillomavirus (HPV) genotypes have been judged to be carcinogenic or probably carcinogenic, and the cause of virtually all cervical cancer worldwide. Other HPV genotypes could possibly be involved. Although the inclusion of possibly carcinogenic HPV genotypes may hurt test specificity, it may indirectly increase the reassurance following a negative HPV test (i.e. the negative predictive value of an HPV test for cervical precancer and cancer). The future of cervical cancer screening in low-resource setting, however, may include once-in-a-lifetime, low-cost and rapid HPV testing. However, the tradeoff of more false positives for greater reassurance may not be acceptable if the local infrastructure cannot manage the screen positives. Now is the time for the community of scientists, doctors, and public health advocates to use the data presented at the 100th International Agency for Research on Cancer monograph meeting to rationally decide the target HPV genotypes for the next generation of HPV tests for use in high-resource and low-resource settings. The implications of including possibly HPV genotypes on HPV test performance, also for guidance on the use of these tests for cervical cancer prevention programs, are discussed

The FANC pathway is activated by adenovirus infection and promotes viral replication-dependent recombination

Deciphering the crosstalk between a host cell and a virus during infection is important not only to better define viral biology but also to improve our understanding of cellular processes. We identified the FANC pathway as a helper of viral replication and recombination by searching for cellular targets that are modified by adenovirus (Ad) infection and are involved in its outcome. This pathway, which is involved in the DNA damage response and checkpoint control, is altered in Fanconi anaemia, a rare cancer predisposition syndrome. We show here that Ad5 infection activates the FANC pathway independent of the classical DNA damage response. Infection with a non-replicating Ad shows that the presence of viral DNA is not sufficient to induce the monoubiquitination of FANCD2 but still activates the DNA damage response coordinated by phospho-NBS1 and phospho-CHK1. E1A expression alone fails to induce FANCD2 monoubiquitination, indicating that a productive viral infection and/or replication is required for FANC pathway activation. Our data indicate that Ad5 infection induces FANCD2 activation to promote its own replication. Specifically, we show that FANCD2 is involved in the recombination process that accompanies viral DNA replication. This study provides evidence of a DNA damage-independent function of the FANC pathway and identifies a cellular system involved in Ad5 recombination

Rescue of replication failure by Fanconi anaemia proteins

Chromosomal aberrations are often associated with incomplete genome duplication, for instance at common fragile sites, or as a consequence of chemical alterations in the DNA template that block replication forks. Studies of the cancer-prone disease Fanconi anaemia (FA) have provided important insights into the resolution of replication problems. The repair of interstrand DNA crosslinks induced by chemotherapy drugs is coupled with DNA replication and controlled by FA proteins. We discuss here the recent discovery of new FA-associated proteins and the development of new tractable repair systems that have dramatically improved our understanding of crosslink repair. We focus also on how FA proteins protect against replication failure in the context of fragile sites and on the identification of reactive metabolites that account for the development of Fanconi anaemia symptoms

Genomic sequencing of colorectal adenocarcinomas identifies a recurrent VTI1A-TCF7L2 fusion

Prior studies have identified recurrent oncogenic mutations in colorectal adenocarcinoma1 and have surveyed exons of protein-coding genes for mutations in 11 affected individuals2,3. Here we report whole-genome sequencing from nine individuals with colorectal cancer, including primary colorectal tumors and matched adjacent non-tumor tissues, at an average of 30.7× and 31.9× coverage, respectively. We identify an average of 75 somatic rearrangements per tumor, including complex networks of translocations between pairs of chromosomes. Eleven rearrangements encode predicted in-frame fusion proteins, including a fusion of VTI1A and TCF7L2 found in 3 out of 97 colorectal cancers. Although TCF7L2 encodes TCF4, which cooperates with β-catenin4 in colorectal carcinogenesis5,6, the fusion lacks the TCF4 β-catenin–binding domain. We found a colorectal carcinoma cell line harboring the fusion gene to be dependent on VTI1A-TCF7L2 for anchorage-independent growth using RNA interference-mediated knockdown. This study shows previously unidentified levels of genomic rearrangements in colorectal carcinoma that can lead to essential gene fusions and other oncogenic events