Molecular patterns of resistance to immune checkpoint blockade in melanoma.

Immune checkpoint blockade (ICB) has improved outcome for patients with metastatic melanoma but not all benefit from treatment. Several immune- and tumor intrinsic features are associated with clinical response at baseline. However, we need to further understand the molecular changes occurring during development of ICB resistance. Here, we collect biopsies from a cohort of 44 patients with melanoma after progression on anti-CTLA4 or anti-PD1 monotherapy. Genetic alterations of antigen presentation and interferon gamma signaling pathways are observed in approximately 25% of ICB resistant cases. Anti-CTLA4 resistant lesions have a sustained immune response, including immune-regulatory features, as suggested by multiplex spatial and T cell receptor (TCR) clonality analyses. One anti-PD1 resistant lesion harbors a distinct immune cell niche, however, anti-PD1 resistant tumors are generally immune poor with non-expanded TCR clones. Such immune poor microenvironments are associated with melanoma cells having a de-differentiated phenotype lacking expression of MHC-I molecules. In addition, anti-PD1 resistant tumors have reduced fractions of PD1+ CD8+ T cells as compared to ICB naïve metastases. Collectively, these data show the complexity of ICB resistance and highlight differences between anti-CTLA4 and anti-PD1 resistance that may underlie differential clinical outcomes of therapy sequence and combination.

Resilience, Positivity and Social Support as Perceived Stress Predictors among University Students.

Since the COVID-19 pandemic, researchers have been trying to identify which personal resources can contribute to minimizing the mental health costs in students incurred due to the restrictions that disrupted safety and predictability in their academic lives. The aim of the study was to verify if and how individual factors (resilience and positivity) and socio-environmental factors (social support and nationality) allow prediction of the level of perceived stress. University students (n = 559) from Poland, Serbia, and Italy were surveyed using the Perceived Stress Scale (PSS-10), the Brief Resilience Scale (BRS), the Positivity Scale (PS), and the Interpersonal Support Evaluation List (ISEL-12). Personal resources-positivity, resilience, and support-were found to be positively interrelated and significantly associated with stress levels. Additionally, gender and nationality differentiated stress levels. A general linear model (GLM) showed that levels of perceived stress are best explained by resilience, positivity, tangible support, and gender. The results obtained can strengthen students' awareness of personal resources and their protective role in maintaining mental health, as well as contribute to the creation of prevention-oriented educational activities. Nationality was not a significant predictor of the level of perceived stress, which highlights the universality of examined predictors among university students from different countries and suggests that interventions aimed at enhancing these resources could benefit students across different cultural contexts.

The Effect of Autogenic Training in a Form of Audio Recording on Sleep Quality and Physiological Stress Reactions of University Athletes-Pilot Study.

Despite the growing popularity of relaxation training, the effectiveness of an autogenic training (AT) as a method of dealing with sleep problems in group of student athletes is unknown. Therefore, this study aimed to fill this gap. University athletes with decreased sleep quality (selected from 209 participants) were randomly assigned to the experimental (EG, n = 11) and control (CG, n = 11) groups similar in terms of sleep quality, age, gender, type of sport discipline and sport experience. During the 14 days dedicated to performing relaxation training in the form of an audio recording, electronic daily logs and actigraphy were used to monitor the athletes' sleep and daily activity. The EG listened to the recording with suggestions based on AT and CG only to the background music. Pre- and post-measurements of sleep quality by means of the Pittsburg Sleep Quality Index (PSQI) and physiological stress reactions by biofeedback device were performed. In EG and CG, the parameters of sleep and daily activity obtained by actigraphy and daily logs as well as physiological indicators of emotional reactivity did not differ. Sleep quality in PSQI significantly increased after AT usage in EG. AT seems to be an effective method for university athletes in improving subjective sleep quality, but further studies are necessary.

Self-reported chronotype and objective sleep timing in university student athletes and non-athletes.

Objective: The aim of this paper was to test how sport participation and chronotype affect objectively measured sleep timing parameters on workdays. Material and Methods: The sample included 82 student athletes and 40 non-athletes who completed three-day wrist actigraphy monitoring and the Polish version of the Morningness-Eveningness Questionnaire. Results: Eveningness predicted later timing of falling asleep and mid-sleep, but not the wake-up time. Student athletes had earlier wake-up time and shorter sleep duration than non-athletes. Discussion: The results support the view that university students suffer insufficient sleep, especially those participating in extensive sport activity.

Co-targeting CDK4/6 and AKT with endocrine therapy prevents progression in CDK4/6 inhibitor and endocrine therapy-resistant breast cancer.

CDK4/6 inhibitors (CDK4/6i) combined with endocrine therapy have shown impressive efficacy in estrogen receptor-positive advanced breast cancer. However, most patients will eventually experience disease progression on this combination, underscoring the need for effective subsequent treatments or better initial therapies. Here, we show that triple inhibition with fulvestrant, CDK4/6i and AKT inhibitor (AKTi) durably impairs growth of breast cancer cells, prevents progression and reduces metastasis of tumor xenografts resistant to CDK4/6i-fulvestrant combination or fulvestrant alone. Importantly, switching from combined fulvestrant and CDK4/6i upon resistance to dual combination with AKTi and fulvestrant does not prevent tumor progression. Furthermore, triple combination with AKTi significantly inhibits growth of patient-derived xenografts resistant to combined CDK4/6i and fulvestrant. Finally, high phospho-AKT levels in metastasis of breast cancer patients treated with a combination of CDK4/6i and endocrine therapy correlates with shorter progression-free survival. Our findings support the clinical development of ER, CDK4/6 and AKT co-targeting strategies following progression on CDK4/6i and endocrine therapy combination, and in tumors exhibiting high phospho-AKT levels, which are associated with worse clinical outcome.

Distinct mechanisms of resistance to fulvestrant treatment dictate level of ER independence and selective response to CDK inhibitors in metastatic breast cancer.

BACKGROUND: Resistance to endocrine treatment in metastatic breast cancer is a major clinical challenge. Clinical tools to predict both drug resistance and possible treatment combination approaches to overcome it are lacking. This unmet need is mainly due to the heterogeneity underlying both the mechanisms involved in resistance development and breast cancer itself. METHODS: To study the complexity of the mechanisms involved in the resistance to the selective estrogen receptor degrader (SERD) fulvestrant, we performed comprehensive biomarker analyses using several in vitro models that recapitulate the heterogeneity of developed resistance. We further corroborated our findings in tissue samples from patients treated with fulvestrant. RESULTS: We found that different in vitro models of fulvestrant resistance show variable stability in their phenotypes, which corresponded with distinct genomic alterations. Notably, the studied models presented adaptation at different cell cycle nodes to facilitate progression through the cell cycle and responded differently to CDK inhibitors. Cyclin E2 overexpression was identified as a biomarker of a persistent fulvestrant-resistant phenotype. Comparison of pre- and post-treatment paired tumor biopsies from patients treated with fulvestrant revealed an upregulation of cyclin E2 upon development of resistance. Moreover, overexpression of this cyclin was found to be a prognostic factor determining resistance to fulvestrant and shorter progression-free survival. CONCLUSIONS: These data highlight the complexity of estrogen receptor positive breast cancer and suggest that the development of diverse resistance mechanisms dictate levels of ER independence and potentially cross-resistance to CDK inhibitors.

Types of Cognitive Appraisal and Undertaken Coping Strategies during Sport Competitions.

The main aim of the research was to distinguish different types of sport competition appraisals and verify if athletes' interpretation of a stressful situation changed their choice of coping methods. Athletes change their perception during competitions; thus, we assumed that configuration of different ways of interpreting stressful events is more important for coping than one particular appraisal. In total, 193 athletes filled out The Stress Appraisal Questionnaire and The Sport Stress-Coping Strategies Questionnaire to describe their stress appraisals and undertaken coping strategies during a remembered competition that took place within a month before the study. The athletes most often appraised stressful competitions as a challenge. They preferred the coping strategy of being determined to accomplish the established goal. The athletes hardly applied techniques that constituted the basis of mental training. The cluster analysis of the competitors determined three types of sport competition appraisals: positive, negative, and active. An ANOVA with post hoc comparisons showed that participants who revealed positive appraisals undertook the highest number of actions aimed at reaching goals and least frequently sought support. Athletes should be taught not only specific strategies for coping with stress, but also more frequent use of positive judgments of sports competitions.

Sleep quality of student athletes and non-athletes - the role of chronotype, stress and life satisfaction.

OBJECTIVE: There are many internal and external factors that can affect sleep deterioration. The adopted model of the relationship between chronotype, stress, life satisfaction and sleep quality was verified in the study. MATERIAL AND METHODS: In total, 335 healthy university students were surveyed using the Morningness-Eveningness Questionnaire, Perceived Stress Scale, Satisfaction with Life Scale and Pittsburgh Sleep Quality Index. The study included two groups: individuals involved in sport activities (student athletes, n=207) and those who declared (in the short form of the International Physical Activity Questionnaire) low physical activity level (non-athlete students, n=128). RESULTS: Student athletes were less stressed (p<0.001) and declared higher life satisfaction (p<0.001) and sleep quality (p<0.001) compared to non-athletes. Non-athletes tended to identify the evening hours as their best time for functioning (p<0.001), but the mean results of both groups oscillated around the so-called intermediate type. Despite the differences in mean values, the model invariance for both groups was confirmed, which means that the proposed theoretical model applies equally to student athletes and non-athletes. The path analysis results indicate that chronotype has a direct negative influence on sleep quality (preferring morning hours results in higher sleep quality). However, perceived stress partially mediates this relationship (p<0.001). DISCUSSION: Sleep quality should not be considered without taking into account circadian preferences. Effective coping with stress may also be a buffer in reducing sleep problems.

Female Volleyball Players Are More Prone to Cortisol Anticipatory Stress Response than Sedentary Women.

Background and Objectives: Top-level sports performance places heavy physical and psychological demands on elite-level athletes, which can be a source of increased levels of stress. Therefore, high-level volleyball players may present altered cardiovascular and endocrinological stress response during stressful events. Although many previous studies have examined the response to stress on athletes, most of them regarded only males, while the impact of the female menstrual cycle has rarely been taken into account. We aimed to study psychophysiological response to anticipatory stressor through analysis of heart rate, self-reported anxiety level, and salivary cortisol in healthy young female athletes by minimalizing the effect of confounders. Materials and Methods: A total of 55 females (25 members of the best league for female volleyball players in Poland and 30 sedentary-lifestyle control subjects) in the follicular phase of their menstrual cycle were exposed to mental arithmetic tasks as an experimental imitation of the stressor. Volleyball players were significantly taller than sedentary individuals (177.1 ± 3.4 cm vs. 173.3 ± 3.4 cm, respectively, p = 0.034), but did not differ in weight (73.6 ± 5.2 kg vs. 70 ± 4.23 kg, respectively, p = 0.081), body mass index (BMI) (23.5 ± 1.13 vs. 24.1 ± 1.45, respectively, p = 0.060), and age (22 ± 1.11 vs. 23 ± 1.14 years, respectively, p = 0.2). Their stress responses were assessed through self-reported anxiety levels and physiological measurements of salivary cortisol concentrations and heart rate (HR). Results: For HR, significant effects of time (F(2,120) = 21.34, p < 0.001, η2 = 0.26) were found, but not for training status (F(1,60) = 2.69, p = 0.106, η2 = 0.04). For cortisol levels, the analysis showed the main effects of time (F(3,180) = 11.73, p < 0.001, η2 = 0.16) and training status (F(1,60) = 4.69, p = 0.034, η2 = 0.07) and a significant interaction between training status and time (F(3,180) = 3.07, p = 0.029, η2 = 0.05). Post-hoc analyses showed higher cortisol concentrations among volleyball players following the math task (all p < 0.001), as well as higher cortisol concentrations in S2, S3, and S4 compared to S1 in volleyball players (all p < 0.001). We observed also a significant increase in state anxiety in both groups (all p < 0.001), but no differences in state anxiety levels between groups. Conclusion: Female volleyball players may not differ in subjective graduation of stressors; however, exposure to training-based stressors seems to promote cortisol response to the anticipated stressor.

Comparison of good and poor sleepers: stress and life satisfaction of university athletes / Comparación de los durmientes Buenos y malos: Estrés y satisfacción de vida de atletas universitarios

The aim of the present study was to compare differences in level of perceived stress, type of stress appraisal and life satisfaction between university athletes who declare problems with sleep (Poor Sleepers, PS, n = 72) and those without problems (Good Sleepers, GS, n = 105). In preliminary analyses the PS and GS were compared for group homogeneity with (a) the Chi-Square test for gender and type of sport, and (b) the Mann-Whitney U test for practice time and weekly frequency of trainings. In both analyses no differences were present. In the main analysis, prepared with the Mann-Whitney U test, GS and PS were compared in terms of psychological variables. Athletes who reveal problems with sleep quality (in comparison to their counterparts with good sleep quality) most often appraise stressful situations as a threat, perceive more stress in everyday life and declare lower life satisfaction. The differences between GS and PS groups may result from the bi-directional relationship between sleep quality and stress and life satisfaction. High stress and low life satisfaction can result in poor sleep quality and, on the other hand, sleep problems can aggravate stress and reduce the life satisfaction. Further studies and the use of advanced analyzes are needed to indicate the relationship between the studied variables in university athletes (e.g., mediation analysis, SEM) (AU)

El objetivo del presente estudio fue comparar las diferencias en el nivel de estrés percibido, el tipo de evaluación del estrés y la satisfacción con la vida entre los atletas universitarios quienes declaran tener problemas de sueño (durmientes malos, DM, n = 72) y aquellos sin tales problemas (durmientes buenos, DB, n = 105). En los análisis preliminares los DM y los DB se compararon por la homogeneidad del grupo sobre la base de (a) la prueba Chi-Square por el género y el tipo de deporte, y (b) la prueba U de Mann-Whitney por el tiempo de práctica y la frecuencia semanal de entrenamientos. En ambos análisis no se detectaron diferencias. En el análisis principal, preparado con la prueba U de Mann-Whitney, los DB y los DM se compararon en términos de variables psicológicas. Los atletas quienes revelan problemas con la calidad del sueño (en comparación con sus homólogos con buena calidad del sueño) suelen evaluar las situaciones estresantes como una amenaza, perciben más estrés en la vida cotidiana y declaran una menor satisfacción con la vida. Las diferencias entre los grupos de DB y DM pueden ser el resultado de la relación bidireccional entre la calidad del sueño y el estrés y la satisfacción con la vida. El alto estrés y la baja satisfacción con la vida pueden resultar en mala calidad del sueño y, por otro lado, los problemas del sueño pueden agravar el estrés y reducir la satisfacción con la vida. Se requieren los estudios posteriores y el uso de análisis avanzados para indicar la relación entre las variables estudiadas en los atletas universitarios (p.ej., análisis de mediación, SEM) (AU)

Indolin-2-one compounds targeting thioredoxin reductase as potential anticancer drug leads.

Several compounds bearing the indolinone chemical scaffold are known to possess anticancer properties. For example, the tyrosine kinase inhibitor sunitinib is an arylideneindolin-2-one compound. The chemical versatility associated with structural modifications of indolinone compounds underlies the potential to discover additional derivatives possessing anticancer properties. Previously synthesized 3-(2-oxoethylidene)indolin-2-one compounds, also known as supercinnamaldehyde (SCA) compounds in reference to the parent compound 1 [1-methyl-3(2-oxopropylidene)indolin-2-one], bear a nitrogen-linked α,ß-unsaturated carbonyl (Michael acceptor) moiety. Here we found that analogs bearing N-substituents, in particular compound 4 and 5 carrying an N-butyl and N-benzyl substituent, respectively, were strongly cytotoxic towards human HCT 116 colorectal and MCF-7 breast carcinoma cells. These compounds also displayed strong thioredoxin reductase (TrxR) inhibitory activity that was likely attributed to the electrophilicity of the Michael acceptor moiety. Their selectivity towards cellular TrxR inhibition over related antioxidant enzymes glutathione reductase (GR), thioredoxin (Trx) and glutathione peroxidase (GPx) was mediated through targeting of the selenocysteine (Sec) residue in the highly accessible C-terminal active site of TrxR. TrxR inhibition mediated by indolin-2-one compounds led to cellular Trx oxidation, increased oxidative stress and activation of apoptosis signal-regulating kinase 1 (ASK1). These events also led to activation of p38 and JNK mitogen-activated protein kinase (MAPK) signaling pathways, and cell death with apoptotic features of PARP cleavage and caspase 3 activation. In conclusion, these results suggest that indolin-2-one-based compounds specifically targeting TrxR may serve as novel drug leads for anticancer therapy.

Thioredoxin-dependent regulation of AIF-mediated DNA damage.

The thioredoxin (Trx) system is one major redox system in mammalian cells. One of its component, Trx, is involved in redox homeostasis and many cellular biological processes through participating in disulfide reduction, S-nitrosylation/S-denitrosylation reactions and protein-protein interactions. In this study, we report the identification of a novel interaction between cytosolic/nuclear Trx1 and apoptosis inducing factor (AIF), and the redox sensitivity and biological significance of the Trx-AIF interaction was characterized. Cytosolic Trx1 but not mitochondrial Trx2 was observed to interact with AIF under physiological conditions and Trx1's active site cysteines were crucial for the interaction. Under oxidative stress conditions, Trx-AIF interaction was disrupted. When the treated cells were allowed to recover from oxidative stress by means of removal of the oxidants, interaction between Trx1 and AIF was re-established time-dependently, which underpins the biological relevance of a Trx-dependent redox regulation of AIF-mediated cell death. Indeed, in times of oxidative stress, nuclear translocation of AIF was found to occur concurrently with perturbations to the Trx-AIF interaction. Once localized in the nucleus, reduced Trx1 hindered the interaction between AIF and DNA, thereby bringing about an attenuation of AIF-mediated DNA damage. In conclusion, characterization of the Trx-AIF interaction has led to an understanding of the effect of reduced Trx1 on possibly regulating AIF-dependent cell death through impeding AIF-mediated DNA damage. Importantly, identification of the novel interaction between Trx1 and AIF has provided opportunities to design and develop therapeutically relevant strategies that either promote or prevent this protein-protein interaction for the treatment of different disease states.

Identification of Michael acceptor-centric pharmacophores with substituents that yield strong thioredoxin reductase inhibitory character correlated to antiproliferative activity.

AIMS: The role of thioredoxin reductase (TrxR) in tumorigenesis has made it an attractive anticancer target. A systematic approach for development of novel compounds as TrxR inhibitors is currently lacking. Structurally diversified TrxR inhibitors share in common electrophilic propensities for the sulfhydryl groups, among which include the Michael reaction acceptors containing an α,ß-unsaturated carbonyl moiety. We aimed to identify features among structurally diversified Michael acceptor-based compounds that would yield a strong TrxR inhibitory character. RESULTS: Structurally dissimilar Michael acceptor-based natural compounds such as isobutylamides, zerumbone, and shogaols (SGs) were found to possess a poor TrxR inhibitory activity, indicating that a sole Michael acceptor moiety was insufficient to produce TrxR inhibition. The 1,7-diphenyl-hept-3-en-5-one pharmacophore in 3-phenyl-3-SG, a novel SG analog that possessed comparable TrxR inhibitory and antiproliferative potencies as 6-SG, was modified to yield 1,5-diphenyl-pent-1-en-3-one (DPPen) and 1,3-diphenyl-pro-1-en-3-one (DPPro, also known as chalcone) pharmacophores. These Michael acceptor-centric pharmacophores, when substituted with the hydroxyl and fluorine groups, gave rise to analogs displaying a TrxR inhibitory character positively correlated to their antiproliferative potencies. Lead analogs 2,2'-diOH-5,5'-diF-DPPen and 2-OH-5-F-DPPro yielded a half-maximal TrxR inhibitory concentration of 9.1 and 10.5 µM, respectively, after 1-h incubation with recombinant rat TrxR, with the C-terminal selenocysteine residue found to be targeted. INNOVATION: Identification of Michael acceptor-centric pharmacophores among diversified compounds demonstrates that a systematic approach to discover and develop Michael acceptor-based TrxR inhibitors is feasible. CONCLUSION: A strong TrxR inhibitory character correlated to the antiproliferative potency is attributed to structural features that include an α,ß-unsaturated carbonyl moiety centered in a DPPen or DPPro pharmacophore bearing hydroxyl and fluorine substitutions.