GDNF/GFRA1 signaling contributes to chemo- and radioresistance in glioblastoma.

Glioblastoma is the most common primary brain tumor in adults, characterized by an inherent aggressivity and resistance to treatment leading to poor prognoses. While some resistance mechanisms have been elucidated, a deeper understanding of these mechanisms is needed to increase therapeutic efficacy. In this study we first discovered glial-cell derived neurotrophic factor (GDNF) to be upregulated in patient-derived glioblastoma spheroid cultures after chemotherapeutic temozolomide treatment, through RNA-Seq experiments. Therefore, we investigated the role of the GDNF/GDNF receptor alpha 1 (GFRA1) signaling pathway as a resistance mechanism to chemotherapy with temozolomide and lomustine, as well as irradiation using patient-derived glioblastoma spheroid cultures. With qPCR experiments we showed a consistent upregulation of GDNF and its primary receptor GFRA1 following all three lines of treatment. Moreover, CRISPR/Cas9 knock-outs of GDNF in two patient-derived models sensitized these cells to chemotherapy treatment, but not radiotherapy. The increased sensitivity was completely reversed by the addition of exogeneous GDNF, confirming the key role of this factor in chemoresistance. Finally, a CRISPR KO of GFRA1 demonstrated a similar increased sensitivity to temozolomide and lomustine treatment, as well as radiotherapy. Together, our findings support the role of the GDNF/GFRA1 signaling pathway in glioblastoma chemo and radioresistance.

How pregnant women with diabetes experience telemonitoring of the fetal heart rate - An interview study.

OBJECTIVES: The use of telemonitoring in healthcare is generally increasing. Women with complicated pregnancies are using telemonitoring as an alternative to conventional management, encompassing hospitalization or frequent outpatient clinic visits. However, there is sparse evidence on how pregnant women experience monitoring of their unborn babies at home. Women might feel uncomfortable with this responsibility, and moreover they might miss face-to-face contact with healthcare personnel. STUDY DESIGN: The study setting was a Danish hospital with a tertiary obstetric unit attending approximately 3400 births annually. A qualitative study design with interview as method included 11 pregnant women with type 1 diabetes or Gestational Diabetes Mellitus. This design was used to investigate how pregnant women with complicated pregnancies experienced telemonitoring of the fetus. Reflexive thematic analysis was used to analyze the pregnant women's experiences of telemonitoring. RESULTS: Women with type 1 diabetes or Gestational Diabetes Mellitus found the advantages of telemonitoring to outweigh the disadvantages. They experienced telemonitoring as time-saving and that telemonitoring decreased the level of stress. Moreover, telemonitoring supports positive collaboration with healthcare professionals. The women also experienced a lack of coordination of consultations between different departments at the hospital and challenges with timing, feedback, and technical issues. Moreover, the women requested an opportunity to discuss family formation and emotions. CONCLUSIONS: Pregnant women with type 1 diabetes or Gestational Diabetes Mellitus benefit from the use of telemonitoring. To further improve the implementation and use of telemonitoring clinical implications, consider how timing and coordination of care, technical equipment, and feedback mechanisms could be improved.

The epidemiology of cryptorchidism and potential risk factors, including endocrine disrupting chemicals.

Congenital cryptorchidism, also known as undescended testis, is the condition where one or both testes are not in place in the scrotum at birth and is one of the most common birth defects in boys. Temporal trends and geographic variation in the prevalence of cryptorchidism from 1% to 9% have been reported in prospective cohort studies. The testes develop in the abdominal cavity and descend to the scrotum in two phases, which should be completed by gestational week 35. Thus, the risk of cryptorchidism is higher in preterm boys. In many cases a spontaneous descent occurs during the first months of life during the surge of gonadotropins and testosterone. If not, the testis is usually brought down to the scrotum, typically by surgery, to increase future fertility chances and facilitate cancer surveillance. The increasing frequency of impaired semen quality and testicular cancer, with which cryptorchidism is associated, represents a concern for male reproductive health in general and a need to understand its risk factors. The risk of cryptorchidism is closely related to gestational factors (preterm birth, low birth weight and intrauterine growth restriction), and especially maternal smoking seems to be a risk factor. Evidence is accumulating that the increasing prevalence of cryptorchidism is also related to prenatal exposure to environmental chemicals, including endocrine disrupting compounds. This association has been corroborated in rodents and supported by ecological studies. Conducting human studies to assess the effect of endocrine disrupting chemicals and their interactions is, however, challenged by the widespread concomitant exposure of all humans to a wide range of chemicals, the combined effect of which and their interactions are highly complex.

Ovarian follicular fluid levels of phthalates and benzophenones in relation to fertility outcomes.

BACKGROUND: Many endocrine disrupting chemicals (EDCs), for instance phthalates and benzophenones, are associated with adverse fertility outcomes and semen quality parameters. OBJECTIVE: To evaluate if concentrations of selected phthalate metabolites and benzophenones measured in follicular fluid are associated with fertility outcomes (i.e., reproductive hormones, antral follicle count, detected heartbeat at gestational week 7, and live birth) and, in a supplementary study, if measured concentrations of chemicals in follicular fluid can exert biological effects on human spermatozoa. METHODS: Overall, 111 couples from a fertility clinic in Denmark contributed with 155 follicular fluid samples. Concentrations of 43 metabolites from 19 phthalates and phthalate substitutes and six benzophenones were measured in follicular fluid using liquid chromatography-tandem mass spectrometry. Multiple linear and logistic regression with an applied generalized estimating equation model allowing more than one measurement per woman assessed the association between follicular EDC levels and fertility outcomes. The assessment of biological effects of individual and mixtures of EDCs on human spermatozoa was conducted through a human sperm cell based Ca2+-fluorimetric assay. RESULTS: Benzophenone-3 (BP-3) and seven metabolites of five phthalates were detectable in follicular fluid. Women with metabolites of dibutyl phthalate isomers in the highest tertiles had lower antral follicle count (MiBP: ß = -5.35 [95 % CI: -9.06; -2.00], MnBP: ß = -5.25 [95 % CI: -9.00; -2.00]) and lower odds for detecting a heartbeat at gestational week 7 (MiBP: OR = 0.35 [95 % CI: 0.14; 0.91], MnBP: OR = 0.39 [95 % CI: 0.13; 1.15]). Mixtures of the measured concentrations of BP-3 and the seven phthalate metabolites induced a small significant increase in the intracellular calcium ion concentration in human spermatozoa from healthy donors (n = 3). DISCUSSION: Phthalate metabolites and BP-3 were detectable in follicular fluid and high concentrations of some phthalate metabolites were linked with lower chance of successful fertility treatment outcomes. Chemical mixture concentrations in follicular fluid induced a calcium response in human spermatozoa highlighting possible biological effects at physiologically relevant concentrations.

Testicular function is associated with cardiometabolic health markers: A cross-sectional study of 2289 young men.

BACKGROUND: Testicular function, including compensated Leydig cell function, has been indicated to be an early marker of morbidity. OBJECTIVE: To study the association of testicular function and markers of metabolic and cardiovascular health in a population of young men. MATERIALS AND METHODS: A cross-sectional study of 2289 men (median age 19 years, 5-95 percentile 18.4-22.2) from the general population examined between 2012 and 2019. Participants answered a questionnaire, had a blood sample drawn for assessment of reproductive hormone levels and health markers (lipids, glycosylated hemoglobin), delivered a semen sample, underwent physical examination including blood pressure measurements, and dual-energy X-ray absorptiometry scan for assessment of body composition. Associations were assessed in both crude and adjusted linear regression analyses. RESULTS: The men were within the normal reference intervals of their age for reproductive and health biomarkers. Compared to the lowest quartile, having luteinizing hormone levels in the highest quartile was associated with higher mean arterial pressure (1.6 [95% confidence interval: 0.8; 2.5] mmHg), cholesterol (0.1 [95% confidence interval: 0.02; 0.18] mmol/L), and total body fat percentage (1.1 [95% confidence interval: 0.4; 1.8] %-points). Higher serum testosterone levels were associated with more advantageous cardiometabolic health markers and higher total sperm count with a healthier body composition and lower glycosylated hemoglobin. DISCUSSION AND CONCLUSION: In this study of young men, unselected regarding reproductive hormones and semen quality, higher luteinizing hormone was associated with cardiovascular risk factors. Higher testosterone and total sperm count were associated with more favorable cardiometabolic indices. Thus, serum reproductive hormones and semen quality may be early appearing biomarkers of cardiovascular health even among young healthy men, which could potentially be useful for preventive initiatives to reduce the excess mortality and morbidity risk among infertile men. However, our study was cross-sectional and cannot determine causation. Future longitudinal studies of reproductive health in young men are warranted.

Electrogenic reaction step and phospholipid translocation pathway of the mammalian P4-ATPase ATP8A2.

ATP8A2 is a mammalian P4-ATPase (flippase) that translocates the negatively charged lipid substrate phosphatidylserine from the exoplasmic leaflet to the cytoplasmic leaflet of cellular membranes. Using an electrophysiological method based on solid supported membranes, we investigated the electrogenicity of specific reaction steps of ATP8A2 and explored a potential phospholipid translocation pathway involving residues with positively charged side chains. Changes to the current signals caused by mutations show that the main electrogenic event occurs in connection with the release of the bound phosphatidylserine to the cytoplasmic leaflet and support the hypothesis that the phospholipid interacts with specific lysine and arginine residues near the cytoplasmic border of the lipid bilayer during the translocation and reorientation required for insertion into the cytoplasmic leaflet.

Prenatal exposure to phenols and benzophenones in relation to markers of male reproductive function in adulthood.

Introduction: Environmental exposure during fetal life may disrupt testicular development. In humans, a limited number of studies have investigated whether these adverse effects persist into adulthood. Using data from a prospective, population-based birth cohort study, The Copenhagen Mother-Child cohort, the objective was to assess if there is an association between fetal exposure to selected phenols and benzophenones and markers of testicular function in adult men. Methods: Pregnant women were recruited in 1997-2001. Their sons were examined clinically at 18-20 years of age, with focus on adult markers of reproductive function (anogenital distance (AGD), semen quality and reproductive hormones). In total, 101 18-20-year-old men were included, whose mothers during pregnancy had a serum sample drawn and analyzed for bisphenol A (BPA) and seven other simple phenols, as well as six benzophenones. To investigate the association between chemical levels (in tertiles, T1-T3) in relation to markers of reproductive function, univariate and multiple linear regression analyses were performed. Results: In fully adjusted analyses, increased levels of luteinizing hormone (LH) were observed with higher fetal exposure to BPA (percentage difference (95%CI)) (T2: 12% (-8%,36%) and T3: 33% (10%,62%), compared to T1) and benzophenone-3 (BP-3) (T2: 21% (-2%,49%), T3: 18% (-4%,45%)), while no clear association was seen to total testosterone (TT). Higher levels of BPA and BP-3 were associated with a lower TT/LH ratio, although only significant for BPA (p-trend=0.01). No associations were seen to AGD or markers of semen quality. Conclusion: In conclusion, high exposure to BPA and BP-3 was associated with a compensated reduced Leydig cell function but no other changes in markers of reproductive health. As maternal levels of BPA and BP-3 were not correlated, separate effects may be at play. Larger studies on long-term reproductive consequences of prenatal exposures are warranted to validate our findings.

Environmental factors in declining human fertility.

A severe decline in child births has occurred over the past half century, which will lead to considerable population declines, particularly in industrialized regions. A crucial question is whether this decline can be explained by economic and behavioural factors alone, as suggested by demographic reports, or to what degree biological factors are also involved. Here, we discuss data suggesting that human reproductive health is deteriorating in industrialized regions. Widespread infertility and the need for assisted reproduction due to poor semen quality and/or oocyte failure are now major health issues. Other indicators of declining reproductive health include a worldwide increasing incidence in testicular cancer among young men and alterations in twinning frequency. There is also evidence of a parallel decline in rates of legal abortions, revealing a deterioration in total conception rates. Subtle alterations in fertility rates were already visible around 1900, and most industrialized regions now have rates below levels required to sustain their populations. We hypothesize that these reproductive health problems are partially linked to increasing human exposures to chemicals originating directly or indirectly from fossil fuels. If the current infertility epidemic is indeed linked to such exposures, decisive regulatory action underpinned by unconventional, interdisciplinary research collaborations will be needed to reverse the trends.

Free testosterone and cardiometabolic parameters in men: comparison of algorithms.

OBJECTIVE: Calculating the free testosterone level has gained increasing interest and different indirect algorithms have been suggested. The objective was to compare free androgen index (FAI), free testosterone estimated using the linear binding model (Vermeulen: cFTV) and the binding framework accounting for allosterically coupled SHBG monomers (Zakharov: cFTZ) in relation to cardiometabolic conditions. DESIGN: A prospective cohort study including 5350 men, aged 30-70 years, participating in population-based surveys (MONICA I-III and Inter99) from 1982 to 2001 and followed until December 2012 with baseline and follow-up information on cardiometabolic parameters and vital status. RESULTS: Using age-standardized hormone levels, FAI was higher among men with baseline cardiometabolic conditions, whereas cFTV and cFTZ levels were lower compared to men without these conditions as also seen for total testosterone. Men in highest quartiles of cFTV or cFTZ had lower risk of developing type 2 diabetes (cFTV: HR = 0.74 (0.49-1.10), cFTZ: HR = 0.59 (0.39-0.91)) than men in lowest quartile. In contrast, men with highest levels of FAI had a 74% (1.17-2.59) increased risk of developing type 2 diabetes compared to men in lowest quartile. CONCLUSION: The association of estimated free testosterone and the studied outcomes differ depending on algorithm used. cFTV and cFTZ showed similar associations to baseline and long-term cardiometabolic parameters. In contrast, an empiric ratio, FAI, showed opposite associations to several of the examined parameters and may reflect limited clinical utility.

Lysates of Methylococcus capsulatus Bath induce a lean-like microbiota, intestinal FoxP3+RORγt+IL-17+ Tregs and improve metabolism.

Interactions between host and gut microbial communities are modulated by diets and play pivotal roles in immunological homeostasis and health. We show that exchanging the protein source in a high fat, high sugar, westernized diet from casein to whole-cell lysates of the non-commensal bacterium Methylococcus capsulatus Bath is sufficient to reverse western diet-induced changes in the gut microbiota to a state resembling that of lean, low fat diet-fed mice, both under mild thermal stress (T22 °C) and at thermoneutrality (T30 °C). Concomitant with microbiota changes, mice fed the Methylococcus-based western diet exhibit improved glucose regulation, reduced body and liver fat, and diminished hepatic immune infiltration. Intake of the Methylococcu-based diet markedly boosts Parabacteroides abundances in a manner depending on adaptive immunity, and upregulates triple positive (Foxp3+RORγt+IL-17+) regulatory T cells in the small and large intestine. Collectively, these data point to the potential for leveraging the use of McB lysates to improve immunometabolic homeostasis.

Using assessment criteria for pesticides to evaluate the endocrine disrupting potential of non-pesticide chemicals: Case butylparaben.

Regulation of chemicals with endocrine disrupting properties depend on the use of the chemical rather than its intrinsic properties. Within the EU, the only criteria currently in place for identifying an endocrine disrupting chemical (EDC) are those developed for biocidal and plant protection products. We argue that ECHA/EFSA guidance for assessing endocrine disrupting properties of biocidal and plant protection products can be applied to all chemicals independent of their intended use. We have assessed the REACH-registered compound butylparaben (CAS 94-36-8), a preservative used primarily in cosmetics. Based on scientific evidence of adverse reproductive effects and endocrine activity, the open literature suggest that butylparaben is an EDC. By applying the ECHA/EFSA guidance for pesticides and biocides, we identify butylparaben as a compound with endocrine disrupting properties. Even though available data is markedly different from that for biocides and pesticides, it was possible to reach this conclusion. More generally, we propose that the ECHA/EFSA guidance can and should be used for identification of EDC regardless of their intended application.

Impact of psychological stress measured in three different scales on testis function: A cross-sectional study of 1362 young men.

BACKGROUND: Studies have reported associations between psychological stress and semen quality, but most have been performed on selected populations using different stress measures. Thus, it is uncertain which stress scale best quantifies the effects of stress on testicular function. OBJECTIVE: To study the association between three different measures of stress and testicular function in young men. MATERIAL AND METHODS: In total, 1362 men (median age 19 years) delivered semen and blood samples. They also answered a questionnaire including information from three stress scales: Stress Symptoms, Stressful Life Events and Perceived Stress. Various statistical analyses for associations between stress and testicular function (semen quality and reproductive hormones) were performed. RESULTS: Perceived Stress was negatively associated with sperm concentration, total count and motility and positively associated with serum FSH. Men with the highest scores (>30 points) had 38% (95% CI 3-84%) lower sperm concentration, 42% (95% CI 5-91%) lower total count and 22% (95% CI 2-32%) lower proportion of motile spermatozoa than men with the lowest scores (0-10 points). For the stress symptoms score, men with highest scores (>95th percentile vs. lower) had lower sperm concentration, total sperm count, motility and serum Inhibin-B/FSH-ratio. Although men with highest stress levels were characterized by an unhealthier lifestyle, adjusting for lifestyle factors did not attenuate results suggesting that the associations between stress and testicular function were not mediated by lifestyle. Stressful Life Events were not associated with testicular function. DISCUSSION AND CONCLUSION: The linear association between Perceived Stress and semen parameters and lack of dose-response association for the other two stress scales indicated that perceived stress was the most sensitive marker of stress affecting semen quality in young men. The lack of associations between Stressful Life Events and testis function confirmed that the perception of stressful events rather than the stressful event per se matters.

Emergency drug kits at the Danish hospital pharmacies: varying management and challenges.

OBJECTIVES: Access to emergency drug kits (EDK) during medical emergencies can be life-saving; however, recent doubts about the quality of the kits have been expressed. Procurements of pharmaceuticals to the five regional authorities in Denmark are serviced by Amgros, a public sector organisation owned by the regions and established to create economies of scale and achieve administrative savings by centralisation. This means that Amgros calls for tenders for the supply of pharmaceuticals to the hospital pharmacies. The Hospital Pharmacy in the North Denmark Region does not currently have an effective method to manage Amgros procurements in relation to EDKs. Thus, the objectives were to explore how quality in the management and packing of EDKs is assured and maintained at different hospital pharmacies in Denmark and how this is affected by Amgros procurements. METHODS: The hospital pharmacies in Denmark were enrolled in a cross-sectional study. Information about the management and challenges of the EDKs was inquired by means of a questionnaire. Responses were analysed by simple statistics. RESULTS: All eight hospital pharmacies in Denmark completed the questionnaire, and the distribution between single-use and reusable packaging was nearly equal. The hospital pharmacies comply with a variation of regulations of which good distribution practice is the most common. Six hospital pharmacies experience challenges with drug replacements in the EDKs and only one hospital pharmacy complies completely with the Amgros procurement. The majority of the hospital pharmacies use parameters such as price of the new drug and potential expense for new packaging in their decision of whether to comply with the Amgros procurement. CONCLUSION: The management of the EDKs varies greatly among the hospital pharmacies in Denmark, and national requirements are therefore encouraged to ensure the quality. The challenges experienced with drug replacements reflect that complying with the Amgros procurement can be troublesome.

Hippo signaling, actin polymerization, and follicle activation in fragmented human ovarian cortex.

The Hippo pathway has been associated with regulation of early follicle growth. Studies of murine ovaries suggest that changes in the actin cytoskeleton, caused by fragmentation, result in inhibition of the Hippo pathway, and in turn, may activate follicle growth. In humans, the connections between fragmentation, the actin cytoskeleton, and follicle activation are yet to be confirmed. In this study, we investigated the impact in vitro fragmentation of a human ovarian cortex on (a) actin polymerization, (b) components of the Hippo pathway, and (c) follicle growth in vivo. The results showed that the ratio between globular and filamentous actin remained unchanged at all timepoints (0, 10, 30, 60, 120, and 240 min) following tissue fragmentation. Neither was the Hippo pathway effector protein YES-associated protein upregulated nor was gene expression of the downstream growth factors CCN2, CCN3, or CCN5 increased at any timepoint in the fragmented cortex. Furthermore, the number of growing follicles was similar in fragmented and intact cortex pieces after 6 weeks' xenotransplantation. However, the total number of surviving follicles was considerably lower in the fragmented cortex compared with intact tissue, suggesting detrimental effects of fragmentation on tissue grafting. These results indicate that fragmentation is likely to be ineffective to activate follicle growth in the human ovarian cortex.

Psychological stress, stressful life events, male factor infertility, and testicular function: a cross-sectional study.

OBJECTIVE: To assess the association between psychological stress and male factor infertility as well as testicular function (semen quality, serum reproductive hormones) and erectile dysfunction. DESIGN: Cross-sectional study. SETTING: University Hospital-based research center. PATIENTS: Men with impaired semen quality were included from infertile couples, and men with naturally conceived pregnant partners were used as a reference population. INTERVENTIONS: Participants completed a questionnaire on health and lifestyle, including a 14-item questionnaire about self-rated psychological stress symptoms and stressful life event (SLEs), had a physical examination performed, delivered a semen sample and had a blood sample drawn. MAIN OUTCOMES: Differences in stress scores (calculated from self-reported stress symptoms) and SLEs between infertile and fertile men were assessed in crude and fully adjusted linear regression models. Secondary outcomes were semen quality, serum reproductive hormones, and erectile dysfunction. RESULTS: Of 423 men, 176 (41.6%) experienced at least one SLE in the 3 months prior to inclusion (50.4%/36.9%: infertile/fertile men, P = .03); ß-coefficient and 95% confidence interval for the difference between the groups on the transformed scale in fully adjusted linear regression models was 0.18 (0.06, 0.30). However, there were no differences in psychological stress symptoms between the two groups (ß-coefficient and 95% confidence interval) on the transformed scale (0.14; -0.02, 0.30). No association between stress (self-reported stress symptoms and SLEs) and testicular function or with erectile dysfunction was found in any of the men. CONCLUSION: Infertile men reported a higher number of SLEs than fertile men but did not report more psychological stress symptoms. Distress and SLEs were not associated with reduced male reproductive function.

Associations of Fish Oil Supplement Use With Testicular Function in Young Men.

Importance: Many young men have poor semen quality, and the causes are often unknown. Supplement intake of ω-3 polyunsaturated fatty acid has been found to improve semen quality among men with infertility, but the association with semen quality among healthy men is unknown. Objective: To determine if intake of ω-3 fatty acid supplements is associated with testicular function as measured by semen quality and reproductive hormone levels among healthy men. Design, Setting, and Participants: This cross-sectional study included young Danish men from the general population recruited between January 1, 2012, and December 31, 2017, at compulsory examinations to determine their fitness for military service. Young unselected men were approached after the examination and invited to participate in a study of reproductive function, regardless of their fitness for military service. Data analysis was conducted from September 1, 2018, to June 30, 2019. Exposures: Intake of supplements, including fish oil, during the past 3 months. Main Outcomes and Measures: Semen quality, measured as volume, concentration, total sperm count, percentage of morphologically normal spermatozoa, and motility, and serum reproductive hormone levels, measured as follicle-stimulating hormone, luteinizing hormone, testosterone, free testosterone, and inhibin B levels. Results: Among 1679 young Danish men (median [interquartile range] age, 18.9 [18.7-19.4] years) recruited to participate, 98 men (5.8%) reported use of fish oil supplements during the past 3 months, of whom 53 (54.1%) reported intake on 60 or more days. After adjustment and compared with men with no supplement intake, men with fish oil supplement intake on fewer than 60 days had semen volume that was 0.38 (95% CI, -0.03 to 0.80) mL higher, and men with fish oil supplement intake on 60 or more days had semen volume that was 0.64 (95% CI, 0.15 to 1.12) mL higher (P for trend < .001). Similarly, testicular size in men with supplement intake on fewer than 60 days was 0.8 (95% CI, -0.2 to 1.9) mL larger and in men with fish oil supplement intake on 60 or more days was 1.5 (95% CI, 0.2 to 2.8) mL larger compared with men with no supplement intake (P for trend = .007). After adjustment, men with fish oil supplement intake had a 20% (95% CI, 9%-31%) lower follicle-stimulating hormone level and 16% (95% CI, 8%-24%) lower luteinizing hormone level compared with men with no supplement intake. There were no associations of intake of other supplements with measures of testicular function. Conclusions and Relevance: These findings suggest that intake of fish oil supplements was associated with better testicular function, which is less likely to be due to confounding by indication, as no associations of intake of other supplements with testicular function were found. This cross-sectional study did not examine the actual content of ω-3 fatty acids in the supplements; therefore, these findings need confirmation in well-designed randomized clinical trials among unselected men.

Truncated PPM1D impairs stem cell response to genotoxic stress and promotes growth of APC-deficient tumors in the mouse colon.

Protein phosphatase magnesium-dependent 1 delta (PPM1D) terminates cell response to genotoxic stress by negatively regulating the tumor suppressor p53 and other targets at chromatin. Mutations in the exon 6 of the PPM1D result in production of a highly stable, C-terminally truncated PPM1D. These gain-of-function PPM1D mutations are present in various human cancers but their role in tumorigenesis remains unresolved. Here we show that truncated PPM1D impairs activation of the cell cycle checkpoints in human non-transformed RPE cells and allows proliferation in the presence of DNA damage. Next, we developed a mouse model by introducing a truncating mutation in the PPM1D locus and tested contribution of the oncogenic PPM1DT allele to colon tumorigenesis. We found that p53 pathway was suppressed in colon stem cells harboring PPM1DT resulting in proliferation advantage under genotoxic stress condition. In addition, truncated PPM1D promoted tumor growth in the colon in Apcmin mice and diminished survival. Moreover, tumor organoids derived from colon of the ApcminPpm1dT/+ mice were less sensitive to 5-fluorouracil when compared to ApcminPpm1d+/+and the sensitivity to 5-fluorouracil was restored by inhibition of PPM1D. Finally, we screened colorectal cancer patients and identified recurrent somatic PPM1D mutations in a fraction of colon adenocarcinomas that are p53 proficient and show defects in mismatch DNA repair. In summary, we provide the first in vivo evidence that truncated PPM1D can promote tumor growth and modulate sensitivity to chemotherapy.

Phosphatidylserine flipping by the P4-ATPase ATP8A2 is electrogenic.

Phospholipid flippases (P4-ATPases) utilize ATP to translocate specific phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of biological membranes, thus generating and maintaining transmembrane lipid asymmetry essential for a variety of cellular processes. P4-ATPases belong to the P-type ATPase protein family, which also encompasses the ion transporting P2-ATPases: Ca2+-ATPase, Na+,K+-ATPase, and H+,K+-ATPase. In comparison with the P2-ATPases, understanding of P4-ATPases is still very limited. The electrogenicity of P4-ATPases has not been explored, and it is not known whether lipid transfer between membrane bilayer leaflets can lead to displacement of charge across the membrane. A related question is whether P4-ATPases countertransport ions or other substrates in the opposite direction, similar to the P2-ATPases. Using an electrophysiological method based on solid supported membranes, we observed the generation of a transient electrical current by the mammalian P4-ATPase ATP8A2 in the presence of ATP and the negatively charged lipid substrate phosphatidylserine, whereas only a diminutive current was generated with the lipid substrate phosphatidylethanolamine, which carries no or little charge under the conditions of the measurement. The current transient seen with phosphatidylserine was abolished by the mutation E198Q, which blocks dephosphorylation. Likewise, mutation I364M, which causes the neurological disorder cerebellar ataxia, mental retardation, and disequilibrium (CAMRQ) syndrome, strongly interfered with the electrogenic lipid translocation. It is concluded that the electrogenicity is associated with a step in the ATPase reaction cycle directly involved in translocation of the lipid. These measurements also showed that no charged substrate is being countertransported, thereby distinguishing the P4-ATPase from P2-ATPases.

Exploratory analyses of consensus molecular subtype-dependent associations of TP53 mutations with immunomodulation and prognosis in colorectal cancer.

BACKGROUND: Accumulating evidence suggests immunomodulatory and context-dependent effects of TP53 mutations in cancer. We performed an exploratory analysis of the transcriptional, immunobiological and prognostic associations of TP53 mutations within the gene expression-based consensus molecular subtypes (CMSs) of colorectal cancer (CRC). MATERIALS AND METHODS: In a single-hospital series of 401 stage I-IV primary CRCs, we sequenced the whole coding region of TP53 and analysed CMS-dependent transcriptional consequences of the mutations by gene expression profiling. Immunomodulatory associations were validated by multiplex, fluorescence-based immunohistochemistry of immune cell markers. Prognostic associations of TP53 mutations were analysed in an aggregated series of 635 patients classified according to CMS, including publicly available data from a French multicentre cohort (GSE39582). RESULTS: TP53 mutations were found in 60% of the CRCs. However, gene set enrichment analyses indicated that their transcriptional consequences varied among the CMSs and were most pronounced in CMS1-immune and CMS4-mesenchymal. Subtype specificity was primarily seen as an upregulation of gene sets reflecting cell cycle progression in CMS4 and a downregulation of T cell activity in CMS1. The subtype-dependent immunomodulatory associations were reinforced by significant depletion of several immune cell populations in mutated tumours compared with wild-type (wt) tumours exclusively in CMS1, including cytotoxic lymphocytes (adjusted p value in CMS1=0.002 and CMS2-4>0.9, Microenvironment Cell Populations (MCP)-counter algorithm). This was validated by immunohistochemistry-based quantification of tumour infiltrating CD8+ cells. Within CMS1, the immunomodulatory association of TP53 mutations was strongest among microsatellite stable (MSS) tumours, and this translated into a propensity for metastatic disease and poor prognostic value of the mutations specifically in the CMS1/MSS subtype (both series overall survival: TP53 mutation vs wt: HR 5.52, p=0.028). CONCLUSIONS: Integration of TP53 mutation status with the CMS framework in primary CRC suggested subtype-dependent immunobiological associations with prognostic and potentially immunotherapeutic implications, warranting independent validation.

Gene expression profiles of CMS2-epithelial/canonical colorectal cancers are largely driven by DNA copy number gains.

About 80% of colorectal cancers (CRCs) have chromosomal instability, which is an integral part of aggressive malignancy development, but the importance of specific copy number aberrations (CNAs) in modulating gene expression, particularly within the framework of clinically relevant molecular subtypes, remains mostly elusive. We performed DNA copy number profiling of 257 stage I-IV primary CRCs and integrative gene expression analysis in 151 microsatellite stable (MSS) tumors, focusing on high-level amplifications and the effect of CNAs on the characteristics of the gene expression-based consensus molecular subtypes (CMS). The results were validated in 323 MSS tumors from TCGA. Novel recurrent high-level amplifications (≥15 additional copies) with a major impact on gene expression were found for TOX3 (16q) at 1.5% frequency, as well as for CCND2 (12p) and ANXA11 (10q) at 1% frequency, in addition to the well-known targets ERBB2 (17q) and MYC (8q). Focal amplifications with ≥15 or ≥5 additional copies of at least one of these regions were associated with a poor overall survival among patients with stage I-III MSS CRCs (multivariable hazard ratio ≥3.2, p ≤ 0.01). All high-level amplifications were focal and had a more consistent relationship with gene expression than lower amplitude and/or broad-range amplifications, suggesting specific targeting during carcinogenesis. Genome-wide, copy number driven gene expression was enriched for pathways characteristic of the CMS2-epithelial/canonical subtype, including DNA repair and cell cycle progression. Furthermore, 50% of upregulated genes in CMS2-epithelial/canonical MSS CRCs were driven by CNAs, an enrichment compared with the other CMS groups, and associated with the stronger correspondence between CNAs and gene expression in malignant epithelial cells than in the cells of the tumor microenvironment (fibroblasts, endothelial cells, leukocytes). In conclusion, we identify novel recurrent amplifications with impact on gene expression in CRC and provide the first evidence that CMS2 may have a stronger copy-number related genetic basis than subtypes more heavily influenced by gene expression signals from the tumor microenvironment.