Clonal dominance defines metastatic dissemination in pancreatic cancer.

Tumors represent ecosystems where subclones compete during tumor growth. While extensively investigated, a comprehensive picture of the interplay of clonal lineages during dissemination is still lacking. Using patient-derived pancreatic cancer cells, we created orthotopically implanted clonal replica tumors to trace clonal dynamics of unperturbed tumor expansion and dissemination. This model revealed the multifaceted nature of tumor growth, with rapid changes in clonal fitness leading to continuous reshuffling of tumor architecture and alternating clonal dominance as a distinct feature of cancer growth. Regarding dissemination, a large fraction of tumor lineages could be found at secondary sites each having distinctive organ growth patterns as well as numerous undescribed behaviors such as abortive colonization. Paired analysis of primary and secondary sites revealed fitness as major contributor to dissemination. From the analysis of pro- and nonmetastatic isogenic subclones, we identified a transcriptomic signature able to identify metastatic cells in human tumors and predict patients' survival.

Manganese Exacerbates Seasonal Health Declines in a Suicidally Breeding Mammal.

Reproductive costs must be balanced with survival to maximize lifetime reproductive rates; however, some organisms invest in a single, suicidal bout of breeding known as semelparity. The northern quoll (Dasyurus hallucatus) is an endangered marsupial in which males, but not females, are semelparous. Northern quolls living near mining sites on Groote Eylandt, Northern Territory, Australia, accumulate manganese (Mn) in their brains, testes, and hair, and elevated Mn impacts motor performance. Whether Mn is associated with other health declines is yet unknown. In the present study we show that male and female northern quolls with higher Mn accumulation had a 20% reduction in immune function and a trend toward reduced cortisol concentrations in hair. The telomere lengths of male quolls did not change pre- to postbreeding, but those with higher Mn levels had longer telomeres; in contrast, the telomeres of females shortened during the breeding season but recovered between the first year and second year of breeding. In addition, the telomeres of quolls that were recaptured declined at significantly higher rates in quolls with higher Mn between prebreeding, breeding, and/or postbreeding seasons. Future research should determine whether changes in cortisol, immune function, or telomere length affect reproductive output or survival-particularly for semelparous males. Environ Toxicol Chem 2024;43:74-86. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

SMARCB1 regulates the hypoxic stress response in sickle cell trait.

Renal medullary carcinoma (RMC) is an aggressive kidney cancer that almost exclusively develops in individuals with sickle cell trait (SCT) and is always characterized by loss of the tumor suppressor SMARCB1. Because renal ischemia induced by red blood cell sickling exacerbates chronic renal medullary hypoxia in vivo, we investigated whether the loss of SMARCB1 confers a survival advantage under the setting of SCT. Hypoxic stress, which naturally occurs within the renal medulla, is elevated under the setting of SCT. Our findings showed that hypoxia-induced SMARCB1 degradation protected renal cells from hypoxic stress. SMARCB1 wild-type renal tumors exhibited lower levels of SMARCB1 and more aggressive growth in mice harboring the SCT mutation in human hemoglobin A (HbA) than in control mice harboring wild-type human HbA. Consistent with established clinical observations, SMARCB1-null renal tumors were refractory to hypoxia-inducing therapeutic inhibition of angiogenesis. Further, reconstitution of SMARCB1 restored renal tumor sensitivity to hypoxic stress in vitro and in vivo. Together, our results demonstrate a physiological role for SMARCB1 degradation in response to hypoxic stress, connect the renal medullary hypoxia induced by SCT with an increased risk of SMARCB1-negative RMC, and shed light into the mechanisms mediating the resistance of SMARCB1-null renal tumors against angiogenesis inhibition therapies.

Ether phospholipids are required for mitochondrial reactive oxygen species homeostasis.

Mitochondria are hubs where bioenergetics, redox homeostasis, and anabolic metabolism pathways integrate through a tightly coordinated flux of metabolites. The contributions of mitochondrial metabolism to tumor growth and therapy resistance are evident, but drugs targeting mitochondrial metabolism have repeatedly failed in the clinic. Our study in pancreatic ductal adenocarcinoma (PDAC) finds that cellular and mitochondrial lipid composition influence cancer cell sensitivity to pharmacological inhibition of electron transport chain complex I. Profiling of patient-derived PDAC models revealed that monounsaturated fatty acids (MUFAs) and MUFA-linked ether phospholipids play a critical role in maintaining ROS homeostasis. We show that ether phospholipids support mitochondrial supercomplex assembly and ROS production; accordingly, blocking de novo ether phospholipid biosynthesis sensitized PDAC cells to complex I inhibition by inducing mitochondrial ROS and lipid peroxidation. These data identify ether phospholipids as a regulator of mitochondrial redox control that contributes to the sensitivity of PDAC cells to complex I inhibition.

Combined KRASG12C and SOS1 inhibition enhances and extends the anti-tumor response in KRASG12C-driven cancers by addressing intrinsic and acquired resistance.

Efforts to improve the anti-tumor response to KRASG12C targeted therapy have benefited from leveraging combination approaches. Here, we compare the anti-tumor response induced by the SOS1-KRAS interaction inhibitor, BI-3406, combined with a KRASG12C inhibitor (KRASG12Ci) to those induced by KRASG12Ci alone or combined with SHP2 or EGFR inhibitors. In lung cancer and colorectal cancer (CRC) models, BI-3406 plus KRASG12Ci induces an anti-tumor response stronger than that observed with KRASG12Ci alone and comparable to those by the other combinations. This enhanced anti-tumor response is associated with a stronger and extended suppression of RAS-MAPK signaling. Importantly, BI-3406 plus KRASG12Ci treatment delays the emergence of acquired adagrasib resistance in both CRC and lung cancer models and is associated with re-establishment of anti-proliferative activity in KRASG12Ci-resistant CRC models. Our findings position KRASG12C plus SOS1 inhibition therapy as a promising strategy for treating both KRASG12C-mutated tumors as well as for addressing acquired resistance to KRASG12Ci.

Complex I inhibitor of oxidative phosphorylation in advanced solid tumors and acute myeloid leukemia: phase I trials.

Although targeting oxidative phosphorylation (OXPHOS) is a rational anticancer strategy, clinical benefit with OXPHOS inhibitors has yet to be achieved. Here we advanced IACS-010759, a highly potent and selective small-molecule complex I inhibitor, into two dose-escalation phase I trials in patients with relapsed/refractory acute myeloid leukemia (NCT02882321, n = 17) and advanced solid tumors (NCT03291938, n = 23). The primary endpoints were safety, tolerability, maximum tolerated dose and recommended phase 2 dose (RP2D) of IACS-010759. The PK, PD, and preliminary antitumor activities of IACS-010759 in patients were also evaluated as secondary endpoints in both clinical trials. IACS-010759 had a narrow therapeutic index with emergent dose-limiting toxicities, including elevated blood lactate and neurotoxicity, which obstructed efforts to maintain target exposure. Consequently no RP2D was established, only modest target inhibition and limited antitumor activity were observed at tolerated doses, and both trials were discontinued. Reverse translational studies in mice demonstrated that IACS-010759 induced behavioral and physiological changes indicative of peripheral neuropathy, which were minimized with the coadministration of a histone deacetylase 6 inhibitor. Additional studies are needed to elucidate the association between OXPHOS inhibition and neurotoxicity, and caution is warranted in the continued development of complex I inhibitors as antitumor agents.

Epithelial memory of inflammation limits tissue damage while promoting pancreatic tumorigenesis.

Inflammation is a major risk factor for pancreatic ductal adenocarcinoma (PDAC). When occurring in the context of pancreatitis, KRAS mutations accelerate tumor development in mouse models. We report that long after its complete resolution, a transient inflammatory event primes pancreatic epithelial cells to subsequent transformation by oncogenic KRAS. Upon recovery from acute inflammation, pancreatic epithelial cells display an enduring adaptive response associated with sustained transcriptional and epigenetic reprogramming. Such adaptation enables the reactivation of acinar-to-ductal metaplasia (ADM) upon subsequent inflammatory events, thereby limiting tissue damage through a rapid decrease of zymogen production. We propose that because activating mutations of KRAS maintain an irreversible ADM, they may be beneficial and under strong positive selection in the context of recurrent pancreatitis.

Medium-Chain Acyl-CoA Dehydrogenase Protects Mitochondria from Lipid Peroxidation in Glioblastoma.

Glioblastoma (GBM) is highly resistant to chemotherapies, immune-based therapies, and targeted inhibitors. To identify novel drug targets, we screened orthotopically implanted, patient-derived glioblastoma sphere-forming cells using an RNAi library to probe essential tumor cell metabolic programs. This identified high dependence on mitochondrial fatty acid metabolism. We focused on medium-chain acyl-CoA dehydrogenase (MCAD), which oxidizes medium-chain fatty acids (MCFA), due to its consistently high score and high expression among models and upregulation in GBM compared with normal brain. Beyond the expected energetics impairment, MCAD depletion in primary GBM models induced an irreversible cascade of detrimental metabolic effects characterized by accumulation of unmetabolized MCFAs, which induced lipid peroxidation and oxidative stress, irreversible mitochondrial damage, and apoptosis. Our data uncover a novel protective role for MCAD to clear lipid molecules that may cause lethal cell damage, suggesting that therapeutic targeting of MCFA catabolism may exploit a key metabolic feature of GBM. SIGNIFICANCE: MCAD exerts a protective role to prevent accumulation of toxic metabolic by-products in glioma cells, actively catabolizing lipid species that would otherwise affect mitochondrial integrity and induce cell death. This work represents a first demonstration of a nonenergetic role for dependence on fatty acid metabolism in cancer.This article is highlighted in the In This Issue feature, p. 2659.

Uncovering the chemistry of C-C bond formation in C-nucleoside biosynthesis: crystal structure of a C-glycoside synthase/PRPP complex.

The enzyme ForT catalyzes C-C bond formation between 5'-phosphoribosyl-1'-pyrophosphate (PRPP) and 4-amino-1H-pyrazole-3,5-dicarboxylate to make a key intermediate in the biosynthesis of formycin A 5'-phosphate by Streptomyces kaniharaensis. We report the 2.5 Å resolution structure of the ForT/PRPP complex and locate active site residues critical for PRPP recognition and catalysis.

PMP-diketopiperazine adducts form at the active site of a PLP dependent enzyme involved in formycin biosynthesis.

ForI is a PLP-dependent enzyme from the biosynthetic pathway of the C-nucleoside antibiotic formycin. Cycloserine is thought to inhibit PLP-dependent enzymes by irreversibly forming a PMP-isoxazole. We now report that ForI forms novel PMP-diketopiperazine derivatives following incubation with both d and l cycloserine. This unexpected result suggests chemical diversity in the chemistry of cycloserine inhibition.

Exposure to artificial light at night increases innate immune activity during development in a precocial bird.

Humans have greatly altered Earth's night-time photic environment via the production of artificial light at night (ALAN; e.g. street lights, car traffic, billboards, lit buildings). ALAN is a problem of growing importance because it may significantly disrupt the seasonal and daily physiological rhythms and behaviors of animals. There has been considerable interest in the impacts of ALAN on health of humans and other animals, but most of this work has centered on adults and we know comparatively little about effects on young animals. We exposed 3-week-old king quail (Excalfactoria chinensis) to a constant overnight blue-light regime for 6 weeks and assessed weekly bactericidal activity of plasma against Escherichia coli - a commonly employed metric of innate immunity in animals. We found that chronic ALAN exposure significantly increased bactericidal activity and that this elevation in immune performance manifested at different developmental time points in males and females. Whether this short-term increase in immune activity can be extended to wild animals, and whether ALAN-mediated increases in immune activity have positive or negative fitness effects, are unknown and will provide interesting avenues for future studies.

The causative effects of corticosterone on innate immunity during the stress response in the House Sparrow, Passer domesticus.

Stress-induced inhibition of innate immune activity has been observed in a variety of wild birds and may increase chances of infection because this activity constitutes the first line of defense against pathogens. We previously reported that the transient elevation of plasma corticosterone (CORT; the primary avian glucocorticoid) that occurs during stress is necessary for stress-induced suppression of natural antibody-mediated, complement-mediated, and bactericidal activity. Here, we further investigated the regulatory role of CORT during this suppression. To this end, we treated House Sparrows (Passer domesticus) with mitotane to block endogenous CORT production, administered CORT at one of three doses (HI: 1.34 mg/kg; LO: 1.00 mg/kg; CON: vehicle), and assessed natural antibody-mediated, complement-mediated, and bactericidal activity during acute stress induced by handling and restraint. Mitotane administration eliminated the endogenous plasma CORT increase that normally takes place during stress, and corticosterone treatment increased plasma CORT to levels similar to those measured in intact birds during acute stress. As predicted, mitotane-treated birds receiving CON injections did not exhibit stress-induced suppression of complement-mediated and bactericidal activity, and CORT administration at both LO and HI doses restored this suppression. Contrary to expectations, mitotane-treated birds receiving CON injections demonstrated stress-induced suppression of natural antibody-mediated activity. Furthermore, CORT administration did not influence this parameter. These results suggest that stress inhibits innate immune activity through both CORT-dependent and CORT-independent mechanisms, but the contribution of these mechanisms can vary. This variation may result from effects of environmental factors, the identity and role of which warrant further research.

Oxidation of the Cyanobactin Precursor Peptide Is Independent of the Leader Peptide and Operates in a Defined Order.

The five-membered nitrogen plus heteroatom rings known as azolines or in their oxidized form as azoles are very common in natural products and drugs. The oxidation of thiazoline to thiazole in the cyanobactin class of natural products is one of the several important transformations that are known to alter the biological properties of the compound. The ordering of the various chemical reactions that occur during cyanobactin biosynthesis is not fully understood. The structure of the flavin-dependent enzyme responsible for the oxidation of multiple thiazolines reveals it contains an additional domain that in other enzymes recognizes linear peptides. We characterize the activity of the enzyme on two substrates: one with a peptide leader and one without. Kinetics and biophysics reveal that the leader on the substrate is not recognized by the enzyme. The enzyme is faster on either substrate than the macrocyclase or protease in vitro. The enzyme has a preferred order of oxidation of multiple thiazolines in the same linear peptide.

Circulating corticosterone levels vary during exposure to anthropogenic stimuli and show weak correlation with behavior across an urban gradient in house finches (Haemorhous mexicanus).

Urban environments are rapidly expanding and presenting animal populations with novel challenges, many of which are thought to be stressors that contribute to low biodiversity. However, studies on stress responses in urban vs rural populations have produced mixed results, and many of these studies use a standard stressor that cannot be replicated in the wild (e.g. restraining an animal in a bag). Pairing physiological and behavioral measurements in response to urban-related stressors improves our understanding of the mechanism underlying animal success in human-dominated landscapes. Here, we examined the physiological stress (plasma corticosterone, CORT) responses of a songbird species (the house finch, Haemorhous mexicanus) to two different anthropogenic stimuli - (1) the presence of a human and (2) a captive environment containing man-made objects. During three field seasons (summer 2012, winter 2014, and winter 2015), we captured birds at six sites along an urban gradient in Phoenix, Arizona, USA and measured plasma CORT levels both before and after each trial. Though CORT levels did increase post-human exposure, though not during exposure to novel environment, indicating only one of the treatments caused a physiological response, baseline or post-trial plasma CORT levels did not differ between finches between urban and rural birds in 2012 or 2014. However, rural birds demonstrated relatively low pre- and post-trial plasma CORT levels during the human-exposure trials in 2015. Furthermore, we found few correlations between behavioral and physiological responses. A significant positive correlation was only detected between activity behavior after human approach and post-trial plasma CORT levels in 2012. Taken together, our results reveal a weak, conditional relationship between stress physiology, behavioral responses, and urbanization in house finches.

Glucocorticoid receptor selectively mediates stress-induced suppression of innate immunity in the House Sparrow, Passer domesticus.

Stress-induced inhibition of innate immune activity is widespread in free-ranging birds, but the mechanisms that are responsible for this inhibition are poorly understood. We previously demonstrated that an increase in plasma corticosterone (CORT), the primary avian glucocorticoid, is necessary for the inhibition of natural antibody- and complement-mediated as well as bactericidal activities to occur during stress. Here we investigated the role of glucocorticoid receptors in stress-induced inhibition of natural antibody- and complement-mediated activities and bactericidal activity within non-genomic (<10 min) and genomic (<120 min) time frames in male House Sparrows, Passer domesticus. Treatment with the selective glucocorticoid receptor antagonist mifepristone (RU486) attenuated stress-induced suppression of natural antibody-mediated activity within 10 min and 120 min of experimental stress. By contrast, this treatment did not influence stress-induced suppression of complement-mediated or bactericidal activity. These results suggest that stress-induced elevated plasma CORT inhibits natural antibody-mediated activity, but not complement-mediated or bactericidal activity, by activating glucocorticoid receptors, and that both non-genomic and genomic mechanisms underlie this activation. Additional research is needed to identify the receptors that regulate inhibitory effects of elevated plasma CORT on complement-mediated and bactericidal activity.

Roles and Mechanistic Bases of Glucocorticoid Regulation of Avian Reproduction.

To maximize fitness, organisms must invest energetic and nutritional resources into developing, activating, and maintaining reproductive physiology and behavior. Corticosterone (CORT), the primary avian glucocorticoid, regulates energetic reserves to meet metabolic demands. At low (baseline) plasma levels, CORT activates avian mineralocorticoid receptors and may stimulate lipid mobilization, foraging activity, and feeding behavior. During stress in birds, elevated plasma CORT also stimulates glucocorticoid receptors and may promote glycemia, lipolysis, and proteolysis. Furthermore, CORT orchestrates physiological and behavioral adjustments to perceived threats. While many avian studies demonstrate effects of CORT on reproduction, few studies have elucidated the mechanisms, including receptor activation and site(s) of action, which underlie these effects. Even fewer studies have investigated how low and elevated plasma CORT regulates energetic reserves to meet the metabolic demands of reproduction. Here, we propose several hypotheses to clarify the direct and indirect effects of CORT on avian reproductive physiology and behavior. In addition, we emphasize the need for new manipulative studies involving alterations of endogenous plasma CORT levels and/or food availability to elucidate how CORT regulates the energetic demands of reproduction.

Corticosterone rapidly suppresses innate immune activity in the house sparrow (Passer domesticus).

Stress-induced effects on innate immune activity in wild birds have been difficult to predict. These difficulties may arise from the frequent assumptions that (1) the stress response influences different components of the immune response similarly, (2) stress-induced effects do not change over the course of the stress response and (3) glucocorticoids are the primary regulators of stress-induced changes of immune activity. We tested the first two assumptions by measuring three components of innate immunity at two times during the stress response in captive adult male house sparrows, Passer domesticus Acute stress resulting from handling and restraint suppressed plasma lytic and microbicidal activity within 10 min and reduced plasma agglutination ability within 120 min. We tested the third assumption by measuring stress-induced effects in sparrows that were pharmacologically adrenalectomized by mitotane administration. Confirming the effectiveness of this treatment, mitotane-treated birds had lower pre-stress plasma CORT than control birds and showed no increase in plasma CORT during acute stress. The innate immune activity of mitotane-treated birds did not decrease during the stress response, but the pre-stress immune activity of these birds did not differ from that of vehicle-treated birds. These results suggest that elevated plasma CORT during stress is primarily responsible for mediating stress-induced suppression of innate immune activity.

Endocrine, metabolic, and behavioral effects of and recovery from acute stress in a free-ranging bird.

Acute stress in vertebrates generally stimulates the hypothalamo-pituitary-adrenal axis and is often associated with multiple metabolic changes, such as increased gluconeogenesis, and with behavioral alterations. Little information is available, especially in free-ranging organisms, on the duration of these reversible effects once animals are no longer exposed to the stressor. To investigate this question, we exposed free-ranging adult male Rufous-winged Sparrows, Peucaea carpalis, in breeding condition to a standard protocol consisting of a social challenge (conspecific song playback) followed with capture and restraint for 30min, after which birds were released on site. Capture and restraint increased plasma corticosterone (CORT) and decreased plasma testosterone (T), glucose (GLU), and uric acid (UA). In birds that we recaptured the next day after exposure to conspecific song playback, plasma CORT and UA levels no longer differed from levels immediately after capture the preceding day. However, plasma T was similar to that measured after stress exposure the preceding day, and plasma GLU was markedly elevated. Thus, exposure to social challenge and acute stress resulted in persistent (⩾24h) parameter-specific effects. In recaptured sparrows, the territorial aggressive response to conspecific song playback, as measured by song rate and the number of flights over the song-broadcasting speakers, did not, however, differ between the first capture and the recapture, suggesting no proximate functional association between plasma T and conspecific territorial aggression. The study is the first in free-ranging birds to report the endocrine, metabolic, and behavioral recovery from the effects of combined social challenge and acute stress.

The seasonal glucocorticoid response of male Rufous-winged Sparrows to acute stress correlates with changes in plasma uric acid, but neither glucose nor testosterone.

We sought to clarify functional relationships between baseline and acute stress-induced changes in plasma levels of the stress hormone corticosterone (CORT) and the reproductive hormone testosterone (T), and those of two main metabolites, uric acid (UA) and glucose (GLU). Acute stress in vertebrates generally stimulates the secretion of glucocorticoids, which in birds is primarily CORT. This stimulation is thought to promote behavioral and metabolic changes, including increased glycemia. However, limited information in free-ranging birds supports the view that acutely elevated plasma CORT stimulates glycemia. Acute stress also often decreases the secretion of reproductive hormones (e.g., T in males), but the role of CORT in this decrease and the contribution of T to the regulation of plasma GLU remain poorly understood. We measured initial (pre-stress) and acute stress-induced plasma CORT and T as well as GLU in adult male Rufous-winged Sparrows, Peucaea carpalis, sampled during the pre-breeding, breeding, post-breeding molt, and non-breeding stages. Stress increased plasma CORT and the magnitude of this increase did not differ across life history stages. The stress-induced elevation of plasma CORT was consistently associated with decreased plasma UA, suggesting a role for CORT in the regulation of plasma UA during stress. During stress plasma GLU either increased (pre-breeding), did not change (breeding), or decreased (molt and non-breeding), and plasma T either decreased (pre-breeding and breeding) or did not change (molt and non-breeding). These data provide only partial support to the hypothesis that CORT secretion during acute stress exerts a hyperglycemic action or is responsible for the observed decrease in plasma T taking place at certain life history stages. They also do not support the hypothesis that rapid changes in plasma T influence glycemia.

Negative energy balance in a male songbird, the Abert's towhee, constrains the testicular endocrine response to luteinizing hormone stimulation.

Energy deficiency can suppress reproductive function in vertebrates. As the orchestrator of reproductive function, endocrine activity of the hypothalamo-pituitary-gonadal (HPG) axis is potentially an important mechanism mediating such effects. Previous experiments in wild-caught birds found inconsistent relationships between energy deficiency and seasonal reproductive function, but these experiments focused on baseline HPG axis activity and none have investigated the responsiveness of this axis to endocrine stimulation. Here, we present data from an experiment in Abert's towhees, Melozone aberti, using gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) challenges to investigate whether energy deficiency modulates the plasma testosterone responsiveness of the HPG axis. Wild-caught birds were either ad libitum fed or energetically constrained via chronic food restriction during photoinduced reproductive development. Energy deficiency did not significantly affect the development of reproductive morphology, the baseline endocrine activity of the HPG axis, or the plasma testosterone response to GnRH challenge. Energy deficiency did, however, decrease the plasma testosterone responsiveness to LH challenge. Collectively, these observations suggest that energy deficiency has direct gonadal effects consisting of a decreased responsiveness to LH stimulation. Our study, therefore, reveals a mechanism by which energy deficiency modulates reproductive function in wild birds in the absence of detectable effects on baseline HPG axis activity.