Context-dependent Effects Research Articles

Abstract 6271: Hormone receptor inhibition as a strategy for radiosensitization of breast cancer

Abstract Purpose: Expression of the androgen receptor (AR) has been identified as a driver of tumor growth in triple negative breast cancers (TNBC), and previous work has nominated AR as a target for radiosensitization. In addition, 70-95% of all estrogen receptor (ER) positive (ER+) breast cancers also have coexpression of AR, suggesting extended utility of AR inhibition in the radiosensitization of these AR+, ER+ tumors. Here we assessed the efficacy of AR inhibition in ER+, AR+ breast cancers to better understand the role of AR signaling across breast cancer models. Further, we also investigated the effect of ER inhibition on radiosensitization of ER+ breast cancer models. Methods: IC50 values were determined for MDV3100 (enzalutamide), ARN-509 (apalutamide), and ODM-201 (darolutamide) in TNBC cell lines (AR+ TNBC: MDA-MB-453, ACC-422, and SUM-185PE, and AR- TNBC: MDA-MB-231) and ER+ breast cancer cell lines (AR+, ER+: ZR-75-1, BT-474, CAMA-1, and AR-, ER+: MCF-7). IC50 values for tamoxifen were determined for ER+ breast cancer cell lines (MCF-7, T47D, ZR-75-1), and ER- (SUM-159) cells. Clonogenic survival assays were performed to assess radiosensitization with ER or AR inhibition with tamoxifen or second generation anti-androgens, respectively, in TNBC and ER+ breast cancer models. Results: AR inhibition with enzalutamide, apalutamide, and darolutamide showed limited single agent growth inhibition efficacy in AR+ TNBC and AR+, ER+ breast cancer cell lines (IC50 > 10 μM). AR inhibition with enzalutamide did not induce radiosensitivity in vitro. In AR+, ER+ CAMA-1 cells, AR blockade with enzalutamide had a radioprotective effect with enhancement ratios (enhR) of 0.76-0.83. No radiosensitization was observed in BT-474 (enhR: 0.92-1.01) or ZR-75-1 cells (enhR: 0.94-1.00). Radiosensitization was also assessed with anti-androgens apalutamide and darolutamide in AR+ breast cancer models. Inhibition of ER with tamoxifen, however, induced radiosensitization in MCF-7 (enhR: 1.14-1.50) and T47D (enhR: 1.33-1.60) cells. No radiosensitization was observed with tamoxifen in ER- SUM-159 cells. Conclusion: Although AR is a mediator of radioresistance in AR+ TNBC, AR inhibition does not provide comparable radiosensitization in AR+, ER+ models and may actually confer a radioprotective effect. In contrast, our results demonstrate ER inhibition is an effective radiosensitizing strategy in ER+ breast cancers, independent of AR status. This work highlights the complexities of androgen and estrogen receptor signaling in AR+, ER+ breast tumors and underscores the necessity for understanding context dependent effects when translating into patients with AR+ breast cancer. Citation Format: Anna R. Michmerhuizen, Amanda Zhang, Rachel Schwartz, Andrea M. Pesch, Benjamin C. Chandler, Cassandra L. Ritter, Meilan Liu, Kari Wilder-Romans, Daniel E. Spratt, Daniel R. Wahl, Shyam Nyati, Lori J. Pierce, Corey Speers. Hormone receptor inhibition as a strategy for radiosensitization of breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6271.

Abstract 4359: Rational development of novel treatment regimens to improve the efficacy of estrogen therapy in anti-estrogen-resistant ER+ breast cancer

Abstract We hypothesize that the viability of estrogen receptor-positive (ER+) breast cancer cells that acquire resistance to anti-estrogens can be controlled through oscillation of ER stimulation and inhibition. Although anti-estrogen therapy has been highly successful in treating ER+ breast cancer, approximately one third of patients experience recurrent disease and progressively develop resistance to all available anti-estrogen therapies. Paradoxically, treatment with estrogens can elicit anti-cancer effects in tumors with acquired resistance to anti-estrogens. However, the clinical use of estrogen therapy is limited by the lack of a known mechanism of action and a predictive marker of response. In this study, we delineate the context-dependent effects of ER overexpression and use these results to rationally develop novel estrogen therapy treatment regimens to improve tumor response. Using cellular models of ER+ breast cancer, we demonstrate that ER overexpression is a mechanism of resistance to estrogen deprivation, resulting in estrogen-independent ER transcriptional activity. However, ER overexpression is also required for therapeutic response to estrogen. Upon treatment with the endogenous estrogen 17b-estradiol, ER overexpression results in hyperactivation of ER transcriptional activity, DNA damage, and subsequent cell death. DNA damage is ER-dependent, and cell death can be abrogated by knockdown of ER. Additionally, the combination of estrogen therapy with poly (ADP-ribose) polymerase (PARP) inhibition enhances DNA damage and cell death. Using a patient-derived xenograft (PDX) model of therapeutic response to 17b-estradiol, we serially profiled ER expression and transcriptional activity during treatment response and the subsequent development of treatment resistance. Tumors from mice treated with 17b-estradiol exhibit a phenotypic shift between sensitivity to estrogen therapy and estrogen deprivation therapy over the course of treatment. Based on these results, we developed novel treatment schedules and regimens to improve the anti-tumor effects of estrogen therapy. We demonstrate that short-term treatment with 17b-estradiol followed by estrogen deprivation can improve long-term anti-tumor effects, as compared to continuous treatment with 17b-estradiol. In conclusion, we demonstrate the differential effects of ER overexpression in estrogen-depleted vs. estrogen-replete environments, and translate this mechanistic understanding into novel treatment regimens to improve response to estrogen therapy. Citation Format: Nicole A. Traphagen, Amanda Jiang, Sarah R. Hosford, Todd W. Miller. Rational development of novel treatment regimens to improve the efficacy of estrogen therapy in anti-estrogen-resistant ER+ breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4359.

The geophagous earthworm Metaphire guillelmi effects on rhizosphere microbial community structure and functioning vary with plant species

The quantity and quality of plant-derived resources are the primary factors regulating soil microbial communities, however, the top-down control of soil fauna on the soil food web is still underexplored. To integrate bottom-up and top-down forces in mediating soil microbial communities, we evaluated six plant species rhizosphere microbial community structure (indicated by multiple PLFA biomarkers) and functioning (indicated by four different enzyme activities) in the presence and absence of seven adult Metaphire guillelmi earthworms. Six plant species with two legume species (Trifolium repens and Medicago sativa), two grass species (Arundo donax and Lolium perenne) and two grain crops (Triticum aestivum and Zea mays) were used varying with resource demand and nutrient acquisition strategies. We found earthworms led to increasing in similarity of rhizosphere microbial community structure across all studied plant species. For example, the variability of the fungi: bacteria and gram-positive: gram-negative ratios across six plant species was reduced in the presence of earthworms. Furthermore, earthworms generally supported enzyme production in acquiring carbon over nitrogen and phosphorus, and enhanced immobilization sink for N in microbial biomass, suggesting earthworms are more beneficial for microbes in acquiring energy as plant roots-produced enzyme mostly targeted on nutrients. In conclusion, our results revealed a context-dependent earthworm effects on microbial community structure and a general pattern of earthworms in shifting enzyme production across different plant species.

Assessing Rhythmic Visual Entrainment and Reinstatement of Brain Oscillations to Modulate Memory Performance.

The human brain’s ability to store information and remember past events is thought to be orchestrated by the synchronization of neuronal oscillations in various frequency bands. A vast amount of research has found that neural oscillations in the theta (∼4–7 Hz) and alpha (∼8–12 Hz) bands play an important role in memory formation. More specifically, it has been suggested that memory performance benefits if the same oscillatory pattern is present during encoding and retrieval. However, the causal relevance of these oscillations is not well understood. Rhythmic sensory stimulation is thought to entrain ongoing brain oscillations and modulate associated functions (e.g., memory formation). In the present study, we used rhythmic visual stimulation at 6 and 10 Hz to experimentally modulate the memory encoding process in a recognition memory task. In addition, we reinstated oscillatory activity from the encoding episode during retrieval, which has been hypothesized to result in memory performance improvements compared to non-reinstated conditions and incongruent reinstatement. Contrary to our hypothesis, we find no effect of neural entrainment during encoding on subsequent memory performance. Likewise, memory retrieval does not benefit from neural reinstatement. The results are discussed with respect to methodological challenges of rhythmic sensory stimulation as a means to alter cognitive processes and induce context-dependent memory effects.

AMPK Activation by Metformin Promotes Survival of Dormant ER+ Breast Cancer Cells.

Despite adjuvant endocrine therapy for patients with estrogen receptor alpha (ER)-positive breast cancer, dormant residual disease can persist for years and eventually cause tumor recurrence. We sought to deduce mechanisms underlying the persistence of dormant cancer cells to identify therapeutic strategies. Mimicking the aromatase inhibitor-induced depletion of estrogen levels used to treat patients, we developed preclinical models of dormancy in ER+ breast cancer induced by estrogen withdrawal in mice. We analyzed tumor xenografts and cultured cancer cells for molecular and cellular responses to estrogen withdrawal and drug treatments. Publicly available clinical breast tumor gene expression datasets were analyzed for responses to neoadjuvant endocrine therapy. Dormant breast cancer cells exhibited upregulated 5' adenosine monophosphate-activated protein kinase (AMPK) levels and activity, and upregulated fatty acid oxidation. While the antidiabetes AMPK-activating drug metformin slowed the estrogen-driven growth of cells and tumors, metformin promoted the persistence of estrogen-deprived cells and tumors through increased mitochondrial respiration driven by fatty acid oxidation. Pharmacologic or genetic inhibition of AMPK or fatty acid oxidation promoted clearance of dormant residual disease, while dietary fat increased tumor cell survival. AMPK has context-dependent effects in cancer, cautioning against the widespread use of an AMPK activator across disease settings. The development of therapeutics targeting fat metabolism is warranted in ER+ breast cancer.

Divergent responses of soil organic carbon to afforestation

Large-scale afforestation is regarded as an effective natural climate solution. However, afforestation-induced changes in soil organic C (SOC) are poorly quantified due to the paucity of large-scale sampling data. Here, we provide the first comprehensive assessment of the afforestation impact on SOC stocks with a pairwise comparative study of samples from 619 control-and-afforested plot pairs in northern China. We found context-dependent effects of afforestation on SOC: afforestation increases SOC density (SOCD) in C-poor soils but decreases SOCD in C-rich soils, especially in deeper soil. Thus, the fixed biomass/SOC ratio assumed in previous studies could overestimate the SOC enhancement by afforestation. By extrapolating the sampling data to the entire region, we estimate that afforestation increased SOC stocks in northern China by only 234.9 ± 9.6 TgC over the last three decades. The study highlights the importance of including pre-afforestation soil properties in models of soil carbon dynamics and carbon sink projections. Forest planting is considered a natural climate solution, but effects on soil carbon are unclear. This study, in northern China, finds that planting forests increases carbon in soils poor in it and vice versa.

Notch signaling at the crossroads of innate and adaptive immunity.

Notch signaling is an evolutionarily conserved cell-to-cell signaling pathway that regulates cellular differentiation and function across multiple tissue types and developmental stages. In this review, we discuss our current understanding of Notch signaling in mammalian innate and adaptive immunity. The importance of Notch signaling is pervasive throughout the immune system, as it elicits lineage and context-dependent effects in a wide repertoire of cells. Although regulation of binary cell fate decisions encompasses many of the functions first ascribed to Notch in the immune system, recent advances in the field have refined and expanded our view of the Notch pathway beyond this initial concept. From establishing T cell identity in the thymus to regulating mature T cell function in the periphery, the Notch pathway is an essential, recurring signal for the T cell lineage. Among B cells, Notch signaling is required for the development and maintenance of marginal zone B cells in the spleen. Emerging roles for Notch signaling in innate and innate-like lineages such as classical dendritic cells and innate lymphoid cells are likewise coming into view. Lastly, we speculate on the molecular underpinnings that shape the activity and versatility of the Notch pathway.

Cholesterol-modifying drugs in COVID-19.

Infection with severe acute respiratory syndrom coronavirus 2 (SARS-CoV-2) is more likely to lead to poor outcomes in the elderly and those with cardiovascular disease, obesity or metabolic syndrome. Here, we consider mechanisms by which dyslipidaemia and the use of cholesterol-modifying drugs could influence the virus–host relationship. Cholesterol is essential for the assembly, replication and infectivity of enveloped virus particles; we highlight several cholesterol-modifying drugs with the potential to alter the SARS-CoV-2 life cycle that could be tested in in vitro and in vivo models. Although cholesterol is an essential component of immune cell membranes, excess levels can dysregulate protective immunity and promote exaggerated pulmonary and systemic inflammatory responses. Statins block the production of multiple sterols, oxysterols and isoprenoids, resulting in a pleiotropic range of context-dependent effects on virus infectivity, immunity and inflammation. We highlight antiviral, immunomodulatory and anti-inflammatory effects of cholesterol-modifying drugs that merit further consideration in the management of SARS-CoV-2 infection.

Characterization of Context-Dependent Effects on Synthetic Promoters.

Understanding the composability of genetic elements is central to synthetic biology. Even for seemingly well-known elements such as a sigma 70 promoter the genetic context-dependent variability of promoter activity remains poorly understood. The lack of understanding of sequence to function results in highly limited de novo design of novel genetic element combinations. To address this issue, we characterized in detail concatenated “stacked” synthetic promoters including varying spacer sequence lengths and compared the transcription strength to the output of the individual promoters. The proxy for promoter activity, the msfGFP synthesis from stacked promoters was consistently lower than expected from the sum of the activities of the single promoters. While the spacer sequence itself had no activity, it drastically affected promoter activities when placed up- or downstream of a promoter. Single promoter-spacer combinations revealed a bivalent effect on msfGFP synthesis. By systematic analysis of promoter and spacer combinations, a semi-empirical correlation was developed to determine the combined activity of stacked promoters.

Manipulation of Prenatal Thyroid Hormones Does Not Affect Growth or Physiology in Nestling Pied Flycatchers

Hormones transferred from mothers to their offspring are thought to be a tool for mothers to prepare their progeny for expected environmental conditions, thus increasing fitness. Thyroid hormones (THs) are crucial across vertebrates for embryonic and postnatal development and metabolism. Yet yolk THs have mostly been ignored in the context of hormone-mediated maternal effects. In addition, the few studies on maternal THs have yielded contrasting results that could be attributed to either species or environmental differences. In this study, we experimentally elevated yolk THs (within the natural range) in a wild population of a migratory passerine, the European pied flycatcher (Ficedula hypoleuca), and assessed the effects on hatching success, nestling survival, growth, and oxidative status (lipid peroxidation, antioxidant enzyme activity, and oxidative balance). We also sought to compare our results with those of a closely related species, the collared flycatcher (Ficedula albicolis), that has strong ecological and life-history similarities with our species. We found no effects of yolk THs on any of the responses measured. We could detect only a weak trend on growth: elevated yolk THs tended to increase growth during the second week after hatching. Our results contradict the findings of previous studies, including those of the collared flycatcher. However, differences in fledging success and nestling growth between both species in the same year suggest a context-dependent influence of the treatment. This study should stimulate more research on maternal effects mediated by THs and their potential context-dependent effects.

Transcriptional consequences of MBD5 disruption in mouse brain and CRISPR-derived neurons

BackgroundMBD5, encoding the methyl-CpG-binding domain 5 protein, has been proposed as a necessary and sufficient driver of the 2q23.1 microdeletion syndrome. De novo missense and protein-truncating variants from exome sequencing studies have directly implicated MBD5 in the etiology of autism spectrum disorder (ASD) and related neurodevelopmental disorders (NDDs). However, little is known concerning the specific function(s) of MBD5.MethodsTo gain insight into the complex interactions associated with alteration of MBD5 in individuals with ASD and related NDDs, we explored the transcriptional landscape of MBD5 haploinsufficiency across multiple mouse brain regions of a heterozygous hypomorphic Mbd5+/GT mouse model, and compared these results to CRISPR-mediated mutations of MBD5 in human iPSC-derived neuronal models.ResultsGene expression analyses across three brain regions from Mbd5+/GT mice showed subtle transcriptional changes, with cortex displaying the most widespread changes following Mbd5 reduction, indicating context-dependent effects. Comparison with MBD5 reduction in human neuronal cells reinforced the context-dependence of gene expression changes due to MBD5 deficiency. Gene co-expression network analyses revealed gene clusters that were associated with reduced MBD5 expression and enriched for terms related to ciliary function.LimitationsThese analyses included a limited number of mouse brain regions and neuronal models, and the effects of the gene knockdown are subtle. As such, these results will not reflect the full extent of MBD5 disruption across human brain regions during early neurodevelopment in ASD, or capture the diverse spectrum of cell-type-specific changes associated with MBD5 alterations.ConclusionsOur study points to modest and context-dependent transcriptional consequences of Mbd5 disruption in the brain. It also suggests a possible link between MBD5 and perturbations in ciliary function, which is an established pathogenic mechanism in developmental disorders and syndromes.

Soy food intake associates with changes in the metabolome and reduced blood pressure in a gut microbiota dependent manner

Background and aimsConsumption of soy foods has been associated with protection against cardiometabolic disease, but the mechanisms are incompletely understood.We hypothesized that habitual soy food consumption associates with gut microbiome composition, metabolite production, and the interaction between diet, microbiota and metabolites. Methods and resultsWe analyzed dietary soy intake, plasma and stool metabolites, and gut microbiome data from two independent cross-sectional samples of healthy US individuals (N = 75 lean or overweight, and N = 29 obese).Habitual soy intake associated with several circulating metabolites. There was a significant interaction between soy intake and gut microbiome composition, as defined by gut enterotype, on metabolites in plasma and stool. Soy consumption associated with reduced systolic blood pressure, but only in a subset of individuals defined by their gut microbiome enterotype, suggesting that responsiveness to soy may be dependent on microbiome composition. Soy intake was associated with differences in specific microbial taxa, including two taxa mapping to genus Dialister and Prevotella which appeared to be suppressed by high soy intake We identified context-dependent effects of these taxa, where presence of Prevotella was associated with higher blood pressure and a worse cardiometabolic profile, but only in the absence of Dialister. ConclusionsThe gut microbiome is an important intermediate in the interplay between dietary soy intake and systemic metabolism. Consumption of soy foods may shape the microbiome by suppressing specific taxa, and may protect against hypertension only in individuals with soy-responsive microbiota. Clinical trials registryNCT02010359 at clinicaltrials.gov.

S30. SCHIZOPHRENIA IN A FISH: EFFECTS OF NMDA ANTAGONISM ON LOCOMOTOR BEHAVIOR AND SOCIAL INTERACTION IN ZEBRAFISH

BackgroundApplying cross-species approaches in animal preclinical studies of psychiatric disorders can minimize the overinterpretations often generated using specific strains or species in animal models of human conditions. Zebrafish is a model organism widely used in neuroscience research as an alternative to rodents, but protocols relevant to the symptoms and neurobiology of schizophrenia have not been fully established and validated in this species. MK-801, an NMDA receptor antagonist, is frequently used to recapitulate schizophrenia symptoms in rodent models. We thus aimed to evaluate the effects of a concentration curve of MK-801 on locomotor activity, social interaction, and neurochemical parameters related to oxidative stress in adult zebrafish.MethodsWild-type male and female zebrafish (50:50 ratio) were randomly allocated to 4 groups: control; 1 µM MK-801; 5 µM MK-801 or 10 µM MK-801 (n = 12). In the locomotor activity test animals were individually and sequentially placed in (1) a beaker with 200 mL of water for 20 min, (2) test aquarium for 30 min to assess basal locomotor activity, (3) beaker with water or MK-801 at the different concentrations for 20 min, and (4) test aquarium for 60 min. Locomotor and exploratory parameters (total distance traveled and upper zone time) were automatically analyzed using ANY-Maze software. In the social interaction test animals were individually exposed to water or MK-801 for 20 min and then placed for 7 min in a tank flanked by two identical tanks either empty or containing 10 zebrafish serving as social stimuli. Distance traveled was automatically tracked while social interaction time was manually and blindly scored using Boris software; only the last 5 minutes were analyzed. Oxidative damage in the brain was assessed by the levels of thiobarbituric acid reactive substances (TBARS). Data were analyzed by ANOVA followed by Tukey post hoc test.ResultsExposure to 5 µM MK-801 decreased the total distance traveled in the locomotor activity test. In the social interaction test, exposure to 5 and 10 µM MK-801 significantly increased the total distance traveled while reduced the time of social interaction with the stimulus animals. No differences in TBARS levels were found between the groups.DiscussionUnlike what is observed in rodents after MK-801 administration, zebrafish did not show hyperlocomotion at the concentrations tested. Other studies point to the context-dependent effect of this drug, as an increase in locomotor activity is only observed when animals are tested in a novel environment. In the social interaction test, on the other hand, animals were tested in a novel context with social stimulus and zebrafish exhibited hyperlocomotion and decreased social interaction, similar to what is observed in rodents. In contrast to chronic animal models and patients with schizophrenia, there were no differences in TBARS levels after acute exposure to MK-801. These preliminary results reinforce that zebrafish is an alternative model organism useful to the study of psychotic disorders. More behavioral and biochemical tests are needed to achieve reproducible tests and models to study multiple schizophrenia domains in zebrafish.

Matrix mechanotransduction mediated by thrombospondin-1/integrin/YAP in the vascular remodeling

The extracellular matrix (ECM) initiates mechanical cues that activate intracellular signaling through matrix-cell interactions. In blood vessels, additional mechanical cues derived from the pulsatile blood flow and pressure play a pivotal role in homeostasis and disease development. Currently, the nature of the cues from the ECM and their interaction with the mechanical microenvironment in large blood vessels to maintain the integrity of the vessel wall are not fully understood. Here, we identified the matricellular protein thrombospondin-1 (Thbs1) as an extracellular mediator of matrix mechanotransduction that acts via integrin αvβ1 to establish focal adhesions and promotes nuclear shuttling of Yes-associated protein (YAP) in response to high strain of cyclic stretch. Thbs1-mediated YAP activation depends on the small GTPase Rap2 and Hippo pathway and is not influenced by alteration of actin fibers. Deletion of Thbs1 in mice inhibited Thbs1/integrin β1/YAP signaling, leading to maladaptive remodeling of the aorta in response to pressure overload and inhibition of neointima formation upon carotid artery ligation, exerting context-dependent effects on the vessel wall. We thus propose a mechanism of matrix mechanotransduction centered on Thbs1, connecting mechanical stimuli to YAP signaling during vascular remodeling in vivo.

Context-dependent effects of relative temperature extremes on bill morphology in a songbird.

Species increasingly face environmental extremes. Morphological responses to changes in average environmental conditions are well documented, but responses to environmental extremes remain poorly understood. We used museum specimens to investigate relationships between a thermoregulatory morphological trait, bird bill surface area (SA) and a measure of short-term relative temperature extremity (RTE), which quantifies the degree that temperature maxima or minima diverge from the 5-year norm. Using a widespread, generalist species, Junco hyemalis, we found that SA exhibited different patterns of association with RTE depending on the overall temperature regime and on precipitation. While thermoregulatory function predicts larger SA at higher RTE, we found this only when the RTE existed in an environmental context that opposed it: atypically cold minimum temperature in a warm climate, or atypically warm maximum temperature in a cool climate. When environmental context amplified the RTE, we found a negative relationship between SA and RTE. We also found that the strength of associations between SA and RTE increased with precipitation. Our results suggest that trait responses to environmental variation may qualitatively differ depending on the overall environmental context, and that environmental change that extremifies already-extreme environments may produce responses that cannot be predicted from observations in less-extreme contexts.

Cell-specific expression of the transcriptional regulator RHAMM provides a timing mechanism that controls appropriate wound re-epithelialization

Prevention of aberrant cutaneous wound repair and appropriate regeneration of an intact and functional integument require the coordinated timing of fibroblast and keratinocyte migration. Here, we identified a mechanism whereby opposing cell-specific motogenic functions of a multifunctional intracellular and extracellular protein, the receptor for hyaluronan-mediated motility (RHAMM), coordinates fibroblast and keratinocyte migration speed and ensures appropriate timing of excisional wound closure. We found that, unlike in WT mice, in Rhamm-null mice, keratinocyte migration initiates prematurely in the excisional wounds, resulting in wounds that have re-surfaced before the formation of normal granulation tissue, leading to a defective epidermal architecture. We also noted aberrant keratinocyte and fibroblast migration in the Rhamm-null mice, indicating that RHAMM suppresses keratinocyte motility but increases fibroblast motility. This cell context-dependent effect resulted from cell-specific regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) activation and expression of a RHAMM target gene encoding matrix metalloprotease 9 (MMP-9). In fibroblasts, RHAMM promoted ERK1/2 activation and MMP-9 expression, whereas in keratinocytes, RHAMM suppressed these activities. In keratinocytes, loss of RHAMM function or expression promoted epidermal growth factor receptor-regulated MMP-9 expression via ERK1/2, which resulted in cleavage of the ectodomain of the RHAMM partner protein CD44 and thereby increased keratinocyte motility. These results identify RHAMM as a key factor that integrates the timing of wound repair by controlling cell migration.

Video context-dependent effects in recognition memory

Abstract The present study investigated how video-dependent recognition is influenced by context load (number of items presented per context), the sequence of context presentation (successive or shuffled presentation of short video contexts), and item cue strength (length of study time). In addition, the effects of background photographs selected from still images of the video contexts were also investigated. The present study includes 7 experiments, in which a total of 287 undergraduates intentionally studied a list of unrelated words. After a filled 5-min retention interval, participants received a recognition test. In the same-context condition, the same video as at study was presented at test, whereas in the different-context condition, new videos were presented at test. With a context load of 1 item, a concordant effect and context dependent-recognition discrimination were found in two video experiments and one background-photograph experiment. Successive presentation of 4-s video contexts produced context-dependent effects in hit rate and in d' with a context load of 6 items and study time of 4 seconds per item, and with a context load of 18 items and study time of 1.33 (4/3) seconds per item. In contrast, no effect was found in the false alarm rate in either condition. Nonetheless, when the context load was 18 and the study time was 4 s per item, no effect was found for the hit rate, false alarm rate, or d'. Shuffled presentation of 4-s video contexts produced a concordant effect but no effect for d' with a context load 6 or 18. Between-participants manipulation produced a context-based mirror effect. The present results imply that video context functions mainly as a global environmental context, and sometimes as a local environmental context.

FGFR1 Oncogenic Activation Reveals an Alternative Cell of Origin of SCLC in Rb1/p53 Mice

SummaryFibroblast growth factor receptor 1 (FGFR1) is frequently amplified in human small-cell lung cancer (SCLC), but its contribution to SCLC and other lung tumors has remained elusive. Here, we assess the tumorigenic capacity of constitutive-active FGFR1 (FGFR1K656E) with concomitant RB and P53 depletion in mouse lung. Our results reveal a context-dependent effect of FGFR1K656E: it impairs SCLC development from CGRPPOS neuroendocrine (NE) cells, which are considered the major cell of origin of SCLC, whereas it promotes SCLC and low-grade NE bronchial lesions from tracheobronchial-basal cells. Moreover, FGFR1K656E induces lung adenocarcinoma (LADC) from most lung cell compartments. However, its expression is not sustained in LADC originating from CGRPPOS cells. Therefore, cell context and tumor stage should be taken into account when considering FGFR1 inhibition as a therapeutic option.

Significance of distinct specifically enriched missense TP53 mutations in prostate cancer.

377 Background: The most common TP53 alterations are missense mutations occurring in the DNA binding domain. The majority of missense p53 mutants (mut-p53) demonstrate oncogenic gain-of-function (GOF) abilities, irrespective of wild-type p53 presence, and thus contribute to a more aggressive disease. In prostate cancer (PCa), characterized by comparatively low overall mutational burden, TP53 is frequently mutated in both primary and advanced disease. Despite significant progress made in the field, detailed mechanisms of GOF in PCa remain undefined due to differing features of p53 mutants. Methods: Analysis of available datasets was performed to assess TP53 mutational status in PCa patient samples and its correlation with the clinical outcome. Using hormone therapy sensitive and CRPC cells, a panel of cell lines was generated to model the two most frequently occurring mutations in the presence or absence of wild-type TP53, as occurs clinically. CHIP-seq, gene expression arrays, and in vitro and in vivo biological assays were performed to interrogate the significance of mut-p53 in PCa. Results: In PCa, missense mutations are significantly associated with decreased progression-free and overall survival. In PCa patient samples these mutations most commonly occur at the R273 residue, demonstrating specific enrichment when compared to other cancers, with R273C alteration being the most frequent. Using our cell panel, CHIP-seq data revealed an expansion of the p53 cistrome upon expression of R273C and R273H mutants in a manner distinct from p53 stabilization in the presence of wt-p53. Moreover, analysis of the TP53 missense mutant-sensitive transcriptomes demonstrated differential gene expression between these mutants, related to the expression of wild-type TP53 in those cells. Finally, R273C and R273H p53 mutants elicited context dependent effects on canonical p53 functions, thereby modulating distinct downstream biological outcomes. Conclusions: These data expand our knowledge of the underlying mechanisms by which distinct gain-of-function p53 mutants affect prostate cancer, and can lead to identification of novel therapeutic targets to improve clinical outcomes in PCa patients harboring these mutations.

Quantifying the Effects of Charge Regulation on Disorder-Order Transitions of Highly Charged Protein Sequences

Charge regulation, which refers to the modulation of charge states of ionizable residues due to shifted pKa values, is an important albeit often overlooked determinant of sequence-ensemble and sequence-structure relationships of intrinsically disordered and intrinsically ordered proteins. We have developed complimentary computational and experimental approaches that account for the complete ensemble of charge states for a given sequence. This combined approach enables the computational calculation of conformational ensembles as a function of pH, and experimental measurement of charge states as a function of pH, allowing us to quantify the effects of charge regulation on archetypal sequences that are predicted to be intrinsically disordered. Here, we present results from a joint experimental and computational analysis of the effects of charge regulation on block-copolypeptides composed entirely of acidic and basic residues, (E4K4)n. These sequences form stable single alpha helices, although they are erroneously predicted to form disordered ensembles if one assumes fixed charge states for the ionizable residues. We employ a novel capillary electrophoresis method for measuring charge as a function of pH. These data, combined with potentiometric analysis, show that glutamate residues have highly shifted pKa values when compared to those of model compounds. This preferential neutralization of glutamate residues that are internal to the block co-polypeptide sequence engenders alpha helix formation by lowering the free energy of desolvation and enabling the favorable propagation of the alpha helix. These findings are readily explained using simulations based on the recently developed q-canonical Monte Carlo sampling method. Our results suggest that sequence-specific charge regulation can engender large-scale conformational transitions and even underlie the driving forces for phase separation. Further, our results highlight the need for refining disorder predictors based on an improved understanding of context dependent charge regulation effects.