Neuroendocrine Development Research Articles

Effects of prenatal food restriction on reproductive function in adult female rats (911.13)

The percentage of adults born low birth weight (LBW) is increasing due to advances in neonatal medicine. Studies in a variety of model organisms indicate that LBW offspring have global perturbations several physiological systems, making it likely that LBW offspring have impaired reproductive physiology. Therefore, we hypothesized that prenatal food restriction that produces LBW offspring would cause female hypothalamic pituitary gonadal (HPG) axis dysfunction as evidenced by differences in hypothalamic gonadotropin releasing hormone (GnRH), estrous cyclicity and age at puberty and reproductive senescence. In this study, pregnant rats were fed ad libitum or 50% of their normal caloric intake from e10 to e21, all pups were cross‐fostered to control dams and litters were culled to 8 pups. Female offspring weight and size from birth through lactation, age at puberty, estrous cyclicity and PND1 hypothalamic GnRH protein expression via western blot. Restricted offspring exhibited LBW (5.6±0.08vs.6.5±0.06, p<0.05). LBW offspring had earlier onset puberty (33.5±0.2vs.38.2±0.2, p<0.01) and significantly longer estrous cycles by PND90 (6.1±0.4vs.4.7±0.3, p<0.05). Although all control offspring had normal estrous cycles at 8 months of age, 80% of restricted offspring had stopped cycling. In addition, hypothalamic GnRH protein expression was decreased at PND1 in LBW offspring (1±0.1vs.0.5±0.1, p<0.05), suggesting that HPG axis dysfunction is present at birth. These data indicate that prenatal food restriction impedes neuroendocrine development and shortens female reproductive lifespan. Further studies are needed to provide a more complete picture of the consequences of LBW for female reproductive physiology.

Within‐person coupling of changes in cortisol, testosterone, and DHEA across the day in adolescents

We comprehensively examined within-person and between-person associations between cortisol and DHEA and cortisol and testosterone across the day. Data are from a sample of 213 adolescents aged 11-16 (M = 13.7, SD = 1.5 years) from the Northeastern US who were oversampled for psychopathology symptoms. Six repeated measures of hormone levels across 3 days were used to test three specific questions of cortisol-DHEA and cortisol-testosterone associations within individuals (coupling) across the day, and one question of cortisol-DHEA and cortisol-testosterone diurnal slopes were associated between adolescents. Results consistently revealed positive cortisol-DHEA and cortisol-testosterone coupling across the day, often more pronounced in girls relative to boys. Cortisol and DHEA slopes were positively associated, whereas cortisol and testosterone were negatively associated between-adolescents. Findings suggest multiple mechanisms and highlight the multifaceted nature of associations of hormone changes during adolescence and importance of considering both axes for between- and within-person aspects of neuroendocrine development.

ERK1 Mediated Epithelial-Mesenchymal Transition and Neuroendocrine Development in Prostate Cancer Cells That Survive High-dose Ionizing Radiation

ERK1 Mediated Epithelial-Mesenchymal Transition and Neuroendocrine Development in Prostate Cancer Cells That Survive High-dose Ionizing Radiation

Paternal deprivation alters play-fighting, serum corticosterone and the expression of hypothalamic vasopressin and oxytocin in juvenile male mandarin voles

Although early paternal deprivation significantly affects offspring behavioral and neuroendocrine development, the link between paternal deprivation and social play behavior remains unclear. Mandarin voles (Microtus mandarinus) are socially monogamous and display bi-paternal care. The present study examined the development of social play in juvenile male mandarin voles and the paternal influence on play-fighting, vasopressin- and oxytocin-immunoreactive neurons and serum corticosterone and testosterone levels. The results show that social play was more pronounced during postnatal days 28-35, differing from the ontogenetic pattern of other forms of social behavior. On postnatal day 35, the peak in play-fighting activity, paternal deprivation reduced boxing/wrestling levels and vasopressin-immunoreactive neurons in the anterior hypothalamus and oxytocin-immunoreactive neurons in the paraventricular nucleus, but increased vasopressin-immunoreactive neurons in the paraventricular nucleus and corticosterone levels. These results suggest that mandarin voles engage in social play according to an inverted U-shaped curve in ontogeny, and paternal deprivation influences the development of offspring play-fighting; hypothalamic vasopressin, oxytocin and serum corticosterone may play a modulatory role in the alteration of play-fighting elicited by paternal deprivation; decreased play-fighting may correlate with depressed vasopressin levels in the anterior hypothalamus.

Sex Differences in Counting and Timing

Sex Differences in Counting and Timing

Signaling of Cytokines is Important in Regulation of GnRH Neurons

Cytokines encompass a broad class of peptides that mediate signals in a broad range of physiological situations including inflammation, infection, and obesity. The cytokine receptor-associated tyrosine kinase, Jak2, is one of the most important proteins mediating cytokine signaling pathway activation. Recently, our group has demonstrated that Jak2 signaling in the gonadotropin-releasing hormone (GnRH) neuron plays a critical role in fertility in males and females, implicating cytokine activation of the neuron in GnRH neuronal development and function. To date, the specific cytokine(s) essential for activating Jak2 during neuroendocrine development are not known. In this article, we review the evidence for the role of several class 1 cytokines in regulating GnRH neuronal development, GnRH secretion, and GnRH expression.

Lung Cancer Signatures in Plasma Based on Proteome Profiling of Mouse Tumor Models

We investigated the potential of in-depth quantitative proteomics to reveal plasma protein signatures that reflect lung tumor biology. We compared plasma protein profiles of four mouse models of lung cancer with profiles of models of pancreatic, ovarian, colon, prostate, and breast cancer and two models of inflammation. A protein signature for Titf1/Nkx2-1, a known lineage-survival oncogene in lung cancer, was found in plasmas of mouse models of lung adenocarcinoma. An EGFR signature was found in plasma of an EGFR mutant model, and a distinct plasma signature related to neuroendocrine development was uncovered in the small-cell lung cancer model. We demonstrate relevance to human lung cancer of the protein signatures identified on the basis of mouse models.

Cancer Biomarkers: Closer to Delivering on their Promise

Taguchi etal. describe, in this issue of Cancer Cell, a quantitative comparative biomarker discovery approach that integrates animal lung cancer models with validation in well-controlled human clinical study sets. This approach overcomes many of the major barriers that have held back the field of cancer biomarkers in the past.

Abstract 3166: Lung cancer bio-signatures in plasma based on the analysis of mouse models

Abstract Genetically engineered mouse models of cancer have been shown to recapitulate many of the molecular and biological features of human cancer. To identify plasma proteins that show altered abundance during lung cancer development, we applied in-depth quantitative proteomic analysis to plasma from three mouse models of lung adenocarcinoma driven by mutant EGFR, Kras or induced by urethane exposure; a mouse model of small cell lung cancer (SCLC); models of pancreatic, ovarian, colon, prostate and breast cancer; and two mouse models of inflammatory disease as confounders. Plasma proteins from mice with disease and age-matched littermate controls were analyzed by mass spectrometry (MS). Whole cell lysates and conditioned media from 12 lung adenocarcinoma cell lines were also analyzed by MS to identify enriched proteins in conditioned media in common with proteins with increased levels in plasmas from lung tumor bearing mice. Proteins corresponding to 3744 non-redundant IPIs (International Protein Index) that represented protein products from 2260 unique genes were quantified in one or more experiments. Unsupervised hierarchical clustering of plasma proteins revealed clustering of the four lung cancer models and the two breast cancer models, suggesting that protein signatures in the blood are organ-type specific. Fifty-eight percent of the elevated proteins in plasmas from mice with lung adenocarcinoma were identified in conditioned media of one or more human lung cell lines. A biosignature based on plasma proteins regulated by the Titf1/Nkx2-1 transcription factor was identified in plasmas from mouse models of lung adenocarcinoma. In addition, protein network analysis showed that many proteins whose abundance is altered in the plasma of the lung cancer mouse model engineered to express mutant human EGFR are associated with EGFR. A plasma protein signature that is enriched in proteins associated with neuroendocrine development was also identified in a SCLC model. Exploratory studies in human lung cancer and control plasmas of candidate markers identified in mouse models yielded altered levels in lung cancer plasmas concordant with findings in mouse models. Our studies of mouse models have identified plasma based signature for lung cancer that reflect developmental and signaling pathways applicable to human lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3166. doi:10.1158/1538-7445.AM2011-3166

Abstract 3992: Roles of ASH1-miR-375 pathway in development of lung cancers with neuroendocrine features

Abstract Lung cancers with neuroendocrine (NE) features are often very aggressive, though the underlying molecular mechanisms have not been clearly elucidated. We previously reported that the basic helix-loop-helix protein, achaete-scute homologue 1 (ASH1/ASCL1), a master regulator of pulmonary neuroendocrine cell development, is crucially involved in the pathogenesis of lung cancers with neuroendocrine features (NE-lung cancers). In the present study, we searched for miRNAs regulated by ASH1 in order to gain insight into the involvement of miRNAs downstream of ASH1 in NE-lung cancer pathogenesis. We consequently found that introduction of ASH1 into A549 cells markedly induced miR-375 and down-regulated miR-200b. Interestingly, these two miRNAs inversely affected cell-cell interaction and cell motility, and they together conferred looser cell attachment and a more motile feature. miR-375 was found to be directly transactivated by ASH1. Global gene expression analysis using miR-375-introduced A549 cells and the subsequent pathway analysis using the Ingenuity IPA software revealed altered gene expressions in several cancer-related pathways (e.g., cellular movement and morphology). Interestingly, we also found that miR-375 alone can induce NE differentiation markers in A549 cells and that miR-375 is required to elicit ASH1-indued NE features. In accordance with these findings in vitro, clear induction of NE features as well as enhanced tumor growth was observed in xenografts of A549 cells stably expressing miR-375. We further found that a transcriptional co-activator YAP1 is most significantly repressed by miR-375 through recognition of its two miR-375 binding sites. A positive correlation between ASH1 and miR-375 as well as a negative correlation between miR-375 and YAP1 were observed in lung cancers, indicating the ASH1-miR-375-YAP1 regulatory pathway is indeed functionally implemented in lung cancer cells. In addition, we found that growth-regulatory functions of YAP1 is cell-lineage dependent in lung cancer cell lines. In conclusion, the present study identified the ASH1-miR-375 axis, providing clues for a better understanding of the molecular and cellular biological roles of ASH1-centered microRNA regulatory networks in the pathogenesis of NE-lung cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3992. doi:10.1158/1538-7445.AM2011-3992

Prenatal PCBs disrupt early neuroendocrine development of the rat hypothalamus

Neonatal exposure to endocrine disrupting chemicals (EDCs) such as polychlorinated biphenyls (PCBs) can interfere with hormone-sensitive developmental processes, including brain sexual differentiation. We hypothesized that disruption of these processes by gestational PCB exposure would be detectable as early as the day after birth (postnatal day (P) 1) through alterations in hypothalamic gene and protein expression. Pregnant Sprague–Dawley rats were injected twice, once each on gestational days 16 and 18, with one of the following: DMSO vehicle; the industrial PCB mixture Aroclor 1221 (A1221); a reconstituted mixture of the three most prevalent congeners found in humans, PCB138, PCB153, and PCB180; or estradiol benzoate (EB). On P1, litter composition, anogenital distance (AGD), and body weight were assessed. Pups were euthanized for immunohistochemistry of estrogen receptor α (ERα) or TUNEL labeling of apoptotic cells or quantitative PCR of 48 selected genes in the preoptic area (POA). We found that treatment with EB or A1221 had a sex-specific effect on developmental apoptosis in the neonatal anteroventral periventricular nucleus (AVPV), a sexually dimorphic hypothalamic region involved in the regulation of reproductive neuroendocrine function. In this region, exposed females had increased numbers of apoptotic nuclei, whereas there was no effect of treatment in males. For ERα, EB treatment increased immunoreactive cell numbers and density in the medial preoptic nucleus (MPN) of both males and females, while A1221 and the PCB mixture had no effect. PCR analysis of gene expression in the POA identified nine genes that were significantly altered by prenatal EDC exposure, in a manner that varied by sex and treatment. These genes included brain-derived neurotrophic factor, GABAB receptors-1 and -2, IGF-1, kisspeptin receptor, NMDA receptor subunits NR2b and NR2c, prodynorphin, and TGFα. Collectively, these results suggest that the disrupted sexual differentiation of the POA by prenatal EDC exposures is already evident as early as the day after birth, effects that may change the trajectory of postnatal development and compromise adult reproductive function.

The α7 nicotinic acetylcholine receptor and the acute stress response: Maternal genotype determines offspring phenotype

α7 Nicotinic acetylcholine receptors (α7nAchRs) modulate immune activation by suppressing production of pro-inflammatory cytokines in peripheral immune cells. α7nAchRs also modulate inhibitory output in the hippocampus, which provides input to key circuits of the HPA axis. Therefore, the α7 nicotinic acetylcholine receptor gene ( CHRNA7) may be associated with cortisol stress response. Polymorphisms in the CHRNA7 promoter decrease its expression and may dampen the cholinergic response, leading to an increase in inflammation. Increased inflammation may change the intrauterine environment, altering neuroendocrine development in the offspring. Maternal CHRNA7 genotype could affect an offspring's HPA regulation via reprogramming in utero. Patients with allergic disorders have a differential cortisol response to stress. This study utilized samples collected from a cohort of 198 adolescents in a previous study of atopic disorders, who demonstrated a disturbance in HPA response associated with atopy. Salivary cortisol samples collected from the adolescents after a series of laboratory procedures and DNA samples collected from the adolescents and their parents were used for further analysis. DNA samples were genotyped for allelic variation in the CHRNA7 promoter. Genetic association analyses with the cortisol levels were performed in the adolescents. Maternal genotype influences were investigated for the CHRNA7 gene. We also included maternal and child atopy diagnosis as covariates in determining cortisol levels and tested for association of CHRNA7 to atopy. Polymorphisms in the CHRNA7 promoter were associated with lower cortisol levels after a small laboratory stress. Our findings also show that although the child's CHRNA7 genotype affects stress response, the maternal genotype has a stronger influence on cortisol release after stress in male offspring. These effects were independent of atopy status.

Fetal stress. Focus on “Effects of acute acidemia on the fetal cardiovascular defense to acute hypoxemia” by Thakor and Giussani

what constitutes fetal stress, and what are the implications of fetal stress in the course of fetal development? Thakor and Giussani ([17][1]) address a fundamental component of the fetal stress response: acidemia and it role as a modulator of the hypothalamus-pituitary-adrenal (HPA) axis

Brain IGF-1 Receptors Control Mammalian Growth and Lifespan through a Neuroendocrine Mechanism

Mutations that decrease insulin-like growth factor (IGF) and growth hormone signaling limit body size and prolong lifespan in mice. In vertebrates, these somatotropic hormones are controlled by the neuroendocrine brain. Hormone-like regulations discovered in nematodes and flies suggest that IGF signals in the nervous system can determine lifespan, but it is unknown whether this applies to higher organisms. Using conditional mutagenesis in the mouse, we show that brain IGF receptors (IGF-1R) efficiently regulate somatotropic development. Partial inactivation of IGF-1R in the embryonic brain selectively inhibited GH and IGF-I pathways after birth. This caused growth retardation, smaller adult size, and metabolic alterations, and led to delayed mortality and longer mean lifespan. Thus, early changes in neuroendocrine development can durably modify the life trajectory in mammals. The underlying mechanism appears to be an adaptive plasticity of somatotropic functions allowing individuals to decelerate growth and preserve resources, and thereby improve fitness in challenging environments. Our results also suggest that tonic somatotropic signaling entails the risk of shortened lifespan.

Molecular regulation of the hypothalamic‐pituitary‐adrenal axis in adult male guinea pigs after prenatal stress at different stages of gestation

Studies in humans and animals have demonstrated that maternal stress during fetal development can lead to altered hypothalamic-pituitary-adrenal (HPA) axis function and behaviour postnatally. We have previously shown adult male guinea pigs that were born to mothers exposed to a stressor during the phase of rapid fetal brain growth (gestational days (GD) 50, 51 and 52; prenatal stress (PS)50) exhibit significantly increased basal plasma cortisol levels. In contrast, male guinea pig offspring whose mothers were exposed to stress later in gestation (GD60, 61 and 62; PS60) exhibited a significantly higher plasma cortisol response to activation of the HPA axis. In the present study, we hypothesized that the endocrine changes in HPA axis function observed in male guinea pig offspring would be reflected by altered molecular regulation of the HPA axis. Corticosteroid receptors in the hippocampus, hypothalamus and pituitary were measured, as well as corticotropin-releasing hormone (CRH), pro-opiomelanocortin (POMC) and adrenal enzymes in the paraventricular nucleus, pituitary and adrenal cortex, respectively, by in situ hybridization and Western blot. PS50 male offspring exhibited a significant reduction in glucocorticoid receptor (GR) mRNA (P <0.01) in the CA3 region of the hippocampus and significantly increased POMC mRNA (P <0.05) in the pituitary, consistent with the increase in basal HPA axis activity observed. In line with elevated activity of the HPA axis, both PS50 and PS60 male offspring exhibited significantly higher steroidogenic factor (SF)-1 (P <0.001) and melanocortin 2 receptor (MC2-R) mRNA (P <0.001) in the adrenal cortex. This study demonstrates that short periods of prenatal stress during critical windows of neuroendocrine development affect the expression of key regulators of HPA axis activity leading to the changes in endocrine function observed in prenatally stressed male offspring. Further, these changes are dependent on the timing of the maternal stressor, a pattern that is emerging in human studies.

Development of ER-α and ER-β expression in the developing ovine brain and pituitary

Fetal neuroendocrine development in late gestation is critical for maintenance of fetal homeostasis, growth, and readiness for birth. We designed the present study to identify the regional patterns of expression of the two main isoforms of the estrogen receptor, ER-α and ER-β, in the developing ovine fetal brain. Fetal (80, 100, 120, 130, and 145 days gestation), neonatal (1 and 7 days), and adult sheep were euthanized and the following tissues were collected: pituitary, hypothalamus, hippocampus, cerebral cortex, and brainstem. Both ER’s are expressed in the ovine brain as early as 80 days gestation, and the expression of both receptors appears to be developmentally regulated. We conclude that both forms of the estrogen receptor are expressed in fetal brain and pituitary throughout the latter half of gestation.

Effects of Perinatal Polychlorinated Biphenyls on Adult Female Rat Reproduction: Development, Reproductive Physiology, and Second Generational Effects1

Perinatal exposures to endocrine-disrupting chemicals, such as polychlorinated biphenyls (PCBs), can cause latent effects on reproductive function. Here, we tested whether PCBs administered during late pregnancy would compromise reproductive physiology in both the fetally exposed female offspring (F1 generation), as well as in their female offspring (F2 generation). Pregnant Sprague-Dawley rats were treated with the PCB mixture, Aroclor 1221 (A1221; 0, 0.1, 1, or 10 mg/kg), on Embryonic Days 16 and 18. Somatic and reproductive development of F1 and their F2 female offspring were monitored, including ages of eye opening, pubertal landmarks, and serum reproductive hormones. The results showed that low doses of A1221 given during this critical period of neuroendocrine development caused differential effects of A1221 on F1 and F2 female rats. In both generations, litter sex ratio was skewed toward females. In the F1 generation, additional effects were found, including a significant alteration of serum LH in the 1 mg/kg A1221 group. The F2 generation showed more profound alterations, particularly with respect to fluctuations in hormones and reproductive tract tissues across the estrous cycle. On proestrus, the day of the preovulatory GnRH/gonadotropin surge, F2 females whose mothers had been exposed perinatally to A1221 exhibited substantially suppressed LH and progesterone concentrations, and correspondingly smaller uterine and ovarian weights on estrus, compared with F2 descendants of control rats. These latter changes suggest a dysregulation of reproductive physiology. Thus, low levels of exposure to PCBs during late fetal development cause significant effects on the maturation and physiology of two generations of female offspring. These findings have implications for reproductive health and fertility of wildlife and humans.

Target Identification Tool

A challenge for functional genomics has been to make meaningful global measurements of the interactions between transcription factors (and cofactors) and DNA. It has been difficult, especially in large genomes, to explicitly map individual binding sites and individual factor-target gene interactions. Johnson et al . (see the Perspective by Fields) have developed a combination of chromatin immunoprecipitation and ultrahigh-throughput sequencing to achieve high specificity and 50-base pair resolution. This approach was used to study regulation by neuron-restrictive silencer factor (NRSF, also known as REST, for repressor element-1 silencing transcription factor) and identify targets of key positive regulators of pancreatic neuroendocrine development. D. S. Johnson, A. Mortazavi, R. M. Myers, B. Wold, Genome-wide mapping of in vivo protein-DNA interactions. Science 316 , 1497-1502 (2007). [Abstract] [Full Text] S. Fields, Site-seeing by sequencing. Science 316 , 1441-1442 (2007). [Summary] [Full Text]

Effects of weaning age on the behavioural and neuroendocrine development of piglets

Adverse early life experiences can permanently “programme” increases in stress reactivity in adulthood. We investigated whether weaning, which involves maternal separation, at different ages, altered subsequent behaviour, physiology and neuroendocrine function in young pigs. The litters of twelve primiparous sows were randomly allocated to weaning at 12 days (W12), 21 days (W21) or at 42 days (W42). Four test daughters from each litter ( n = 48, 16 from each treatment) were studied from 11 days of age until 90 days of age. At 90 days of age, prior to culling, half of each treatment were challenged with restraint and isolation stress. There was a long-term reduction in salivary cortisol levels following weaning in all groups. W12 and W21 piglets gained weight more slowly after weaning, but by 90 days weights were similar in all groups. Behaviourally, W12 piglets showed more substrate directed and aggressive behaviours, postural changes, and nosing and belly nosing littermates. Surprisingly, W42 piglets exhibited the onset of belly nosing at 28 days while still in the presence of the sow. At 90 days of age there was no evidence that weaning age had persisting effects on the hypothalamic–pituitary–adrenal (HPA) axis. Restraint stress increased plasma ACTH and cortisol levels and CRH mRNA in the hypothalamus demonstrating that the absence of weaning age effects on HPA function was not due to a ceiling effect. This study is the first to investigate long-term effects of weaning age in pigs using a combination of behavioural, physiological and neuroendocrine measures. The results suggest that variation in the age of weaning does not apparently programme the HPA axis but has potentially deleterious effects on behaviour certainly up to 56 days of age. However, our study also raises complications when considering the welfare implications of weaning age. Specifically more information is needed on the causes and long term implications of the sustained elevations of salivary cortisol we observed over the suckling period. The occurrence of belly nosing in later weaned pigs also suggests that care is required when designing environments where sows and piglets are housed together beyond 28 days.

Basic Helix-Loop-Helix Transcription Factor Profiling of Lung Tumors Shows Aberrant Expression of the Proneural Gene Atonal Homolog 1 (ATOH1, HATH1, MATH1) in Neuroendocrine Tumors

Microarray-based expression profiling studies of lung adenocarcinomas have defined neuroendocrine subclasses with poor prognosis. As neuroendocrine development is regulated by members of the achaete-scute and atonal classes of basic helix-loop-helix (bHLH) transcription factors, we analyzed lung tumors for expression of these factors. Out of 13 bHLH genes tested, 4 genes, i.e., achaete-scute complex-like 1 (ASCL1, HASH1, Mash1), atonal homolog 1 (ATOH1, HATH1, MATH1), NEUROD4 (ATH-3, Atoh3, MATH-3) and neurogenic differentiation factor 1 (NEUROD1, NEUROD, BETA2), showed differential expression among lung tumors and absent or low expression in normal lung. As expected, tumors that have high levels of ASCL1 also express neuroendocrine markers, and we found that this is accompanied by increased levels of NEUROD1. In addition, we found ATOH1 expression in 9 (16%) out of 56 analyzed adenocarcinomas and these tumors showed neuroendocrine features as shown by dopa decarboxylase mRNA expression and immunostaining for neuroendocrine markers. ATOH1 expression as well as NEUROD4 was observed in small cell lung carcinoma (SCLC), a known neuroendocrine tumor. Since ATOH1 is not known to be involved in normal lung development, our results suggest that aberrant activation of ATOH1 leads to a neuroendocrine phenotype similar to what is observed for ASCL1 activation during normal neuroendocrine development and in lung malignancies. Our preliminary data indicate that patients with ATOH1-expressing adenocarcinomas might have a worse prognosis.