Biology subjects

Lewis, R. L.

Publications and source records attributed to Lewis, R. L..

8 recordsLinked to original sources

bioRxiv2025

The Utility of Fibroblast Co-culture for the Maintenance of Episomes in Human Papillomavirus-Associated Cancer Models

Human papillomavirus-associated head and neck squamous cell carcinomas (HPV+ HNSCCs) lack early diagnostics and continue to rise in incidence. HPV16 has been detected in [~]90% of HPV+ oropharyngeal cancers (HPV+ OPCs), an anatomical subset of HNSCC, with the majority retaining episomal viral genomes. Despite this, existing episomal HPV16+ OPC cell lines are especially scarce. UMSCC104s were initially reported as episomal; however, the literature contains conflicting reports regarding the genome status of these cells. We now show that UMSCC104s rapidly undergo integration and E2 loss under standard monoculture, and later lots are fully integrated. These findings resolve prior discrepancies and underscore the instability of episomes in monoculture. Accurate models of HPV-driven cancers are critically needed. We propose fibroblast co-culture methods, traditionally utilized for HPV+ keratinocyte models, as a strategy to preserve episomal status in cancer models by supporting viral and host genome stability.

cancer biology↗

bioRxiv2025

HPV16 recruitment of SMARCAL1 to viral and host replication forks is required for the viral life cycle

High-risk human papillomaviruses (HR HPVs) are responsible for around 5% of the worlds cancer burden. Activation and interaction with the host DNA damage response (DDR) promotes the HPV16 life cycle. This study demonstrates a crucial interaction between HPV16 and SMARCAL1, a protein involved in the stabilization of stalled DNA replication forks. SMARCAL1 can complex with E2, is recruited to E1-E2 replicating DNA, and SMARCAL1 knockdown reduces the fidelity of E1-E2 mediated DNA replication in C33a cells but does not alter replication levels. SMARCAL1 is recruited to the HPV16 genome in HPV16-immortalized foreskin keratinocytes (HFK+HPV16), and in situ protein interaction with nascent DNA replication forks (SIRF) assays demonstrated that SMARCAL1 is hyper-recruited to host replication forks in HFK+HPV16 cells. Using COMET, DNA fiber, and cell growth assays it was determined that knockdown of SMARCAL1 increased DNA damage and impaired replication fork progression in HFK+HPV16 cells, ultimately resulting in growth arrest. The viral genome integrates following SMARCAL1 knockdown in HFK+HPV16 cells. Therefore, SMARCAL1 facilitates host and viral DNA replication in HFK+HPV16 cells. Overall, the results demonstrate that HPV16 promotes SMARCAL1 recruitment to viral and host replication forks and is an essential factor for the HPV16 life cycle. The results expand our understanding of DDR proteins that regulate the HPV16 life cycle, and suggest that inhibition of SMARCAL1 function represents a novel anti-viral strategy for the treatment and prevention of HPV infections. ImportanceHPV16 is responsible for the majority of HPV+ cancers, contributing to 54% of cervical cancers and [~]90% of HPV+HNSCC. Integration of viral genomes into host DNA can promote cervical cancer progression and correlates with poor prognosis in HPV-associated HNSCC, where around 70% of HPV+ cancers contain episomal viral genomes. Developing effective antiviral therapies requires a deeper understanding of the interplay between viral replication and host DNA damage response (DDR) pathways. This report demonstrates that SMARCAL1 is essential for HPV16 replication and keratinocyte proliferation and that its depletion leads to replication stress, DNA damage, and viral genome integration. This work underscores the delicate balance between viral exploitation of the host DDR and the risk of genome instability. These insights contribute to the broader understanding of HPV pathogenesis and may inform the development of therapeutic strategies targeting viral replication to prevent disease progression and improve clinical outcomes in HPV-associated cancers.

microbiology↗

bioRxiv2024

Fibroblasts Regulate the Transformation Potential of Human Papillomavirus-positive Keratinocytes

Persistent human papillomavirus (HPV) infection is necessary but insufficient for viral oncogenesis. Additional contributing co-factors, such as immune evasion and viral integration have been implicated in HPV-induced cancer progression. It is widely accepted that HPV+ keratinocytes require co-culture with fibroblasts to maintain viral episome expression, yet the exact mechanisms for this have yet to be elucidated. Here we present comprehensive RNA sequencing and proteomic analysis demonstrating that fibroblasts not only support the viral life cycle, but reduce HPV+ keratinocyte transformation. Our co-culture models offer novel insights into HPV-related transformation mechanisms. HighlightsO_LIFibroblasts support HPV RNA expression and episomal maintenance in HPV+ keratinocytes C_LIO_LIFibroblasts reduce EMT related expression in HPV+ keratinocytes C_LIO_LIFibroblasts promote EMT related expression in E6E7+ keratinocytes C_LI

cancer biology↗

bioRxiv2024

Fibroblast Stromal Support Model for Predicting Human Papillomavirus-Associated Cancer Drug Responses.

Currently, there are no specific antiviral therapeutic approaches targeting Human papillomaviruses (HPVs), which cause around 5% of all human cancers. Specific antiviral reagents are particularly needed for HPV-related oropharyngeal cancers (HPV+OPCs) whose incidence is increasing and for which there are no early diagnostic tools available. We and others have demonstrated that the estrogen receptor alpha (ER) is overexpressed in HPV+OPCs, compared to HPV-negative cancers in this region, and that these elevated levels are associated with an improved disease outcome. Utilizing this HPV+ specific overexpression profile, we previously demonstrated that estrogen attenuates the growth and cell viability of HPV+ keratinocytes and HPV+ cancer cells in vitro. Expansion of this work in vivo failed to replicate this sensitization. The role of stromal support from the tumor microenvironment (TME) has previously been tied to both the HPV lifecycle and in vivo therapeutic responses. Our investigations revealed that in vitro co-culture with fibroblasts attenuated HPV+ specific estrogen growth responses. Continuing to monopolize on the HPV+ specific overexpression of ER, our co-culture models then assessed the suitability of the selective estrogen receptor modulators (SERMs), raloxifene and tamoxifen, and showed growth attenuation in a variety of our models to one or both of these drugs in vitro. Utilization of these SERMs in vivo closely resembled the sensitization predicted by our co-culture models. Therefore, the in vitro fibroblast co-culture model better predicts in vivo responses. We propose that utilization of our co-culture in vitro model can accelerate cancer therapeutic drug discovery. ImportanceHuman papillomavirus-related cancers (HPV+ cancers) remain a significant public health concern, and specific clinical approaches are desperately needed. In translating drug response data from in vitro to in vivo, the fibroblasts of the adjacent stromal support network play a key role. Our study presents the utilization of a fibroblast 2D co-culture system to better predict translational drug assessments for HPV+ cancers. We also suggest that this co-culture system should be considered for other translational approaches. Predicting even a portion of treatment paradigms that may fail in vivo with a co-culture model will yield significant time, effort, resource, and cost efficiencies.

cancer biology↗

bioRxiv2023

Human Papillomavirus 16 replication converts SAMHD1 into a homologous recombination factor and promotes its recruitment to replicating viral DNA

We have demonstrated that SAMHD1 (sterile alpha motif and histidine-aspartic domain HD-containing protein 1) is a restriction factor for the HPV16 life cycle. Here we demonstrate that in HPV negative cervical cancer C33a cells and human foreskin keratinocytes immortalized by HPV16 (HFK+HPV16), SAMHD1 is recruited to E1-E2 replicating DNA. Homologous recombination (HR) factors are required for HPV16 replication and viral replication promotes phosphorylation of SAMHD1, which converts it from a dNTPase to an HR factor independent from E6/E7 expression. A SAMHD1 phosphor-mimic (SAMHD1 T592D) reduces E1-E2 mediated DNA replication in C33a cells and has enhanced recruitment to the replicating DNA. In HFK+HPV16 cells SAMHD1 T592D is recruited to the viral DNA and attenuates cellular growth, but does not attenuate growth in isogenic HFK cells immortalized by E6/E7 alone. SAMHD1 T592D also attenuates the development of viral replication foci following keratinocyte differentiation. The results indicated that enhanced SAMHD1 phosphorylation could be therapeutically beneficial in cells with HPV16 replicating genomes. Protein phosphatase 2A (PP2A) can dephosphorylate SAMHD1 and PP2A function can be inhibited by endothall. We demonstrate that endothall reduces E1-E2 replication and promotes SAMHD1 recruitment to E1-E2 replicating DNA, mimicking the SAMHD1 T592D phenotypes. Finally, we demonstrate that in head and neck cancer cell lines with HPV16 episomal genomes endothall attenuates their growth and promotes recruitment of SAMHD1 to the viral genome. The results suggest that targeting cellular phosphatases has therapeutic potential for the treatment of HPV infections and cancers. ImportanceHuman papillomaviruses are causative agents in around 5% of all human cancers. The development of anti-viral therapeutics depends upon an increased understanding of the viral life cycle. Here we demonstrate that HPV16 replication converts SAMHD1 into an HR factor via phosphorylation. This phosphorylation promotes recruitment of SAMHD1 to viral DNA to assist with replication. A SAMHD1 mutant that mimics phosphorylation is hyper-recruited to viral DNA and attenuates viral replication. Expression of this mutant in HPV16 immortalized cells attenuates the growth of these cells, but not cells immortalized by the viral oncogenes E6/E7 alone. Finally, we demonstrate that the phosphatase inhibitor endothall promotes hyper-recruitment of endogenous SAMHD1 to HPV16 replicating DNA and can attenuate the growth of both HPV16 immortalized human foreskin keratinocytes and HPV16 positive head and neck cancer cell lines. We propose that phosphatase inhibitors represent a novel tool for combating HPV infections and disease.

cancer biology↗

bioRxiv2022

Effects of neural noise on predictive model updating across the adult lifespan

AbstractIn the perceptual and sensorimotor domains, ageing is accompanied by a stronger reliance on top-down predictive model information and reduced sensory learning, thus promoting simpler, more efficient internal models in older adults. Here, we demonstrate analogous effects in higher-order language processing. One-hundred and twenty adults ranging in age from 18 to 83 years listened to short auditory passages containing manipulations of adjective order, with order probabilities varying between two speakers. As a measure of model adaptation, we examined attunement of the N400 event-related potential, a measure of precision-weighted prediction errors in language, to a trial-by-trial measure of speaker-based adjective order expectedness ("speaker-based surprisal") across the course of the experiment. Adaptation was strongest for young adults, weaker for middle-aged adults, and absent for older adults. Over and above age-related differences, we observed individual differences in model adaptation, with aperiodic (1/f) slope and intercept metrics derived from resting-state EEG showing the most pronounced modulations. We suggest that age-related changes in aperiodic slope, which have been linked to neural noise, may be associated with individual differences in the magnitude of stimulus-related prediction error signals. By contrast, changes in aperiodic intercept, which reflects aggregate population spiking, may relate to an individuals updating of inferences regarding stimulus precision. These two mechanisms jointly contribute to age-related changes in the precision-weighting of prediction errors and the degree of sensory learning.

neuroscience↗

bioRxiv2019

Rational use of Episodic and Working Memory: A Normative Account of Prospective Memory

AO_SCPLOWBSTRACTC_SCPLOWHumans often simultaneously pursue multiple plans at different time scales. The successful realization of non-immediate plans (e.g., post package after work) requires keeping track of a future plan while accomplishing other intermediate tasks (e.g., write a paper), a capacity known as prospective memory. This capacity requires the integration of noisy evidence from perceptual input with evidence from short-term working memory (WM) and longer-term or episodic memory (LTM/EM). Here we formulate a set of dual-task problems in empirical studies of prospective memory as problems of computational rationality, and ask how a rational model should exploit noisy perception and memory to maximize payoffs. The model combines reinforcement learning (optimal action selection) with evidence accumulation (optimal inference) in order to derive good decision parameters for optimal task performance (i.e., performing an ongoing task while monitoring for a cue that triggers executing a second prospective task). We compare model behavior to key accuracy and reaction time phenomena in human performance. Thus, we offer a normative approach to theorizing and modeling these phenomena without assumptions about mechanisms of attention or retrieval. This approach can be extended to study meta-parameters governing the boundedly rational use of memory in planned action in health, as well as compensatory mnemonic strategies that may be rational responses to disturbances of these mechanisms in neuropsychiatric disorders.

animal behavior and cognition↗

bioRxiv2018

Human perception-motor decision-making is not optimal

The rationality of human behavior has been a major problem in philosophy for centuries. The pioneering work of Kahneman and Tversky provides strong evidence that people are not rational. Recent work in psychophysics argues that incentivized sensorimotor decisions (such as deciding where to reach to get a reward) maximizes expected gain, suggesting that it may be impervious to cognitive biases and heuristics. We rigorously tested this hypothesis using multiple experiments and multiple computational models. We obtained strong evidence that people deviated from the objectively rational strategy when potential losses were large. They instead appeared to follow a strategy in which they simplify the decision problem and satisfice rather than optimize. This work is consistent with the framework known as bounded rationality, according to which people behave rationally given their computational limitations.

animal behavior and cognition↗