Vagus nerve stimulation does not alter brainstem nuclei morphology in patients with refractory epilepsy.

OBJECTIVE: To describe morphological characteristics of the brainstem nuclei in response to chronic vagus nerve stimulation (VNS) in patients with refractory epilepsy. BACKGROUND: VNS is a treatment option for individuals with medically refractory epilepsy. While treatment with VNS may achieve up to 50% seizure reduction and is protective against sudden unexpected death in epilepsy (SUDEP), its mechanism of action is not fully understood. Long-term structural and cellular changes in response to VNS have rarely been addressed in humans. METHODS: Four autopsy cases with history of chronic epilepsy treated with VNS (VNS+) and 4 age- and sex-matched chronic epilepsy-related death cases without VNS (VNS-) were included. Detailed clinical and postmortem data were obtained. Serial horizontal sections of the brainstem were prepared and stained with hematoxylin, eosin, and luxol fast blue (HE/LFB). Three regions of interest (ROIs) were delineated, including nucleus tractus solitarius (NTS), locus coeruleus (LC), and the rostral pontine group of raphe nuclei (rRN). Immunohistochemistry studies were performed using antibodies to GFAP, NeuN, HLA-DR, and IBA-1. Immunolabeling index was analyzed. RESULTS: Three of the 4 VNS+ patients and all 4 control (VNS-) patients died of SUDEP. There was no laterality difference in the NeuN, GFAP, HLA-DR and IBA-1 expression in LC and NTS of VNS+ patients. Similarly, there was no difference in the rRN, LC, and NTS between the VNS+ and VNS- groups. CONCLUSION: This study represents the first histopathological study of the long-term effects of VNS therapy in the human brain. There was no difference observed in the neuronal cell number, degree of astrocytosis, and neuroinflammation in the main brainstem vagal afferent nuclei after prolonged VNS treatment in patients with refractory epilepsy.

Transcriptome-wide quantitative profiling of PUS7-dependent pseudouridylation by nanopore direct long read RNA sequencing.

Pseudouridylation is a prevalent post-transcriptional RNA modification that impacts many aspects of RNA biology and function. The conversion of uridine to pseudouridine (Ψ) is catalyzed by the family of pseudouridine synthases (PUSs). Development of robust methods to determine PUS-dependent regulation of Ψ location and stoichiometry in low abundant mRNA is essential for biological and functional understanding of pseudouridylation. Here, we present a framework, NanoPsiPy, for identifying Ψ sites and quantify their levels in poly-A RNA at single-nucleotide resolution using direct RNA long-read Nanopore sequencing, based on the observation that Ψ can cause characteristic U-to-C basecalling errors in Nanopore direct RNA sequencing data. Our method was able to detect low and high stoichiometric Ψ sites in human mRNA. We validated our method by transcriptome-wide quantitative profiling of PUS7-dependent Ψ sites in poly-A RNA from a MYCN -amplified neuroblastoma cell line. We identified 8,625 PUS7-dependent Ψ sites in 1,246 mRNAs that encode proteins involved primarily in ribosome biogenesis, translation, and mitochondrial energy metabolism. Our work provides the first example of using direct RNA long-read Nanopore sequencing for transcriptome-wide quantitative profiling of mRNA pseudouridylation regulated by a PUS. We envision that our method will facilitate functional interrogation of PUSs in biological and pathological processes.

HOXC9 directly regulates distinct sets of genes to coordinate diverse cellular processes during neuronal differentiation.

BACKGROUND: Cellular differentiation is characterized by the acquisition of specialized structures and functions, cell cycle exit, and global attenuation of the DNA damage response. It is largely unknown how these diverse cellular events are coordinated at the molecular level during differentiation. We addressed this question in a model system of neuroblastoma cell differentiation induced by HOXC9. RESULTS: We conducted a genome-wide analysis of the HOXC9-induced neuronal differentiation program. Microarray gene expression profiling revealed that HOXC9-induced differentiation was associated with transcriptional regulation of 2,370 genes, characterized by global upregulation of neuronal genes and downregulation of cell cycle and DNA repair genes. Remarkably, genome-wide mapping by ChIP-seq demonstrated that HOXC9 bound to 40% of these genes, including a large number of genes involved in neuronal differentiation, cell cycle progression and the DNA damage response. Moreover, we showed that HOXC9 interacted with the transcriptional repressor E2F6 and recruited it to the promoters of cell cycle genes for repressing their expression. CONCLUSIONS: Our results demonstrate that HOXC9 coordinates diverse cellular processes associated with differentiation by directly activating and repressing the transcription of distinct sets of genes.

Epilepsy-associated death in the Southwestern Ontario: A clinicopathological correlation study.

Patients with epilepsy are at elevated risk for premature mortality, of which sudden unexpected death in epilepsy (SUDEP) is one of the leading causes. SUDEP incidence varies significantly depending on the population and the methods used to document the cause of death. We performed retrospective case review at the London Health Sciences Centre for the period of 2000 to 2018. Clinical information, scene investigations, general pathology findings, toxicology, and neuropathology findings were obtained, examined, and confirmed by two neuropathologists and one epileptologist. The characteristics were compared and summarized. We also evaluated the impact of 2010 revision of Ontario Coroner Act Regulation, which significantly limited whole brain examination. Among the 12,206 cases reviewed, we identified 152 cases with a known history of epilepsy. Ninety-seven cases (64%) were classified as SUDEP. There were significantly more SUDEP decedents found dead unwitnessed at night in prone position, than non-SUDEP. Generalized seizures were strongly associated with SUDEP. A male predominance was observed in SUDEP group between 15 and 35 years old. Near half of the brains examined were "unremarkable." There was no difference in neuropathology findings between SUDEP and non-SUDEP groups. After implementation of the 2010 revision of Ontario Coroner Act Regulation, fixed whole brain examination was reduced from 88% to 7% of the epilepsy-related death investigation. Except a lower diagnosis rate of "inflammatory/infectious changes," there were no significant differences in neuropathology findings. This is the first detailed clinical-pathological study on epilepsy-related death based on a Canadian cohort. This study reinforces the previously reported findings in SUDEP and highlights the importance of clinicopathological correlation for accurate classification of epilepsy-related death.

Caffeine Supplementation and FOXM1 Inhibition Enhance the Antitumor Effect of Statins in Neuroblastoma.

High-risk neuroblastoma exhibits transcriptional activation of the mevalonate pathway that produces cholesterol and nonsterol isoprenoids. A better understanding of how this metabolic reprogramming contributes to neuroblastoma development could help identify potential prevention and treatment strategies. Here, we report that both the cholesterol and nonsterol geranylgeranyl-pyrophosphate branches of the mevalonate pathway are critical to sustain neuroblastoma cell growth. Blocking the mevalonate pathway by simvastatin, a cholesterol-lowering drug, impeded neuroblastoma growth in neuroblastoma cell line xenograft, patient-derived xenograft (PDX), and TH-MYCN transgenic mouse models. Transcriptional profiling revealed that the mevalonate pathway was required to maintain the FOXM1-mediated transcriptional program that drives mitosis. High FOXM1 expression contributed to statin resistance and led to a therapeutic vulnerability to the combination of simvastatin and FOXM1 inhibition. Furthermore, caffeine synergized with simvastatin to inhibit the growth of neuroblastoma cells and PDX tumors by blocking statin-induced feedback activation of the mevalonate pathway. This function of caffeine depended on its activity as an adenosine receptor antagonist, and the A2A adenosine receptor antagonist istradefylline, an add-on drug for Parkinson's disease, could recapitulate the synergistic effect of caffeine with simvastatin. This study reveals that the FOXM1-mediated mitotic program is a molecular statin target in cancer and identifies classes of agents for maximizing the therapeutic efficacy of statins, with implications for treatment of high-risk neuroblastoma. SIGNIFICANCE: Caffeine treatment and FOXM1 inhibition can both enhance the antitumor effect of statins by blocking the molecular and metabolic processes that confer statin resistance, indicating potential combination therapeutic strategies for neuroblastoma. See related commentary by Stouth et al., p. 2091.

NF-κB2 mutation targets survival, proliferation and differentiation pathways in the pathogenesis of plasma cell tumors.

BACKGROUND: Abnormal NF-κB2 activation has been implicated in the pathogenesis of multiple myeloma, a cancer of plasma cells. However, a causal role for aberrant NF-κB2 signaling in the development of plasma cell tumors has not been established. Also unclear is the molecular mechanism that drives the tumorigenic process. We investigated these questions by using a transgenic mouse model with lymphocyte-targeted expression of p80HT, a lymphoma-associated NF-κB2 mutant, and human multiple myeloma cell lines. METHODS: We conducted a detailed histopathological characterization of lymphomas developed in p80HT transgenic mice and microarray gene expression profiling of p80HT B cells with the goal of identifying genes that drive plasma cell tumor development. We further verified the significance of our findings in human multiple myeloma cell lines. RESULTS: Approximately 40% of p80HT mice showed elevated levels of monoclonal immunoglobulin (M-protein) in the serum and developed plasma cell tumors. Some of these mice displayed key features of human multiple myeloma with accumulation of plasma cells in the bone marrow, osteolytic bone lesions and/or diffuse osteoporosis. Gene expression profiling of B cells from M-protein-positive p80HT mice revealed aberrant expression of genes known to be important in the pathogenesis of multiple myeloma, including cyclin D1, cyclin D2, Blimp1, survivin, IL-10 and IL-15. In vitro assays demonstrated a critical role of Stat3, a key downstream component of IL-10 signaling, in the survival of human multiple myeloma cells. CONCLUSIONS: These findings provide a mouse model for human multiple myeloma with aberrant NF-κB2 activation and suggest a molecular mechanism for NF-κB2 signaling in the pathogenesis of plasma cell tumors by coordinated regulation of plasma cell generation, proliferation and survival.

Live tubal ectopic pregnancy managed with localised injection of methotrexate into gestational sac.

Ectopic pregnancy is a pregnancy outside the uterine cavity and is, in majority of cases, a non-viable pregnancy. There are multiple methods of managing patients with ectopic pregnancy including expectant, medical and surgical management. Live tubal ectopic pregnancies, also known as ectopic pregnancies present in the fallopian tube with fetal heartbeat still present, are most commonly treated via surgical route. This case outlines the presentation and an unusual method of management of a patient diagnosed with a live tubal ectopic pregnancy with extensive medical and surgical history.

A Negative Regulatory Role for RKIP in Breast Cancer Immune Response.

Raf-1 kinase inhibitor protein was first identified as a negative regulator of the Raf signaling pathway. Subsequently, it was shown to have a causal role in containing cancer progression and metastasis. Early studies suggested that RKIP blocks cancer progression by inhibiting the Raf-1 pathway. However, it is not clear if the RKIP tumor and metastasis suppression function involve other targets. In addition to the Raf signaling pathway, RKIP has been found to modulate several other signaling pathways, affecting diverse biological functions including immune response. Recent advances in medicine have identified both positive and negative roles of immune response in cancer initiation, progression and metastasis. It is possible that one way that RKIP exerts its effect on cancer is by targeting an immune response mechanism. Here, we provide evidence supporting the causal role of tumor and metastasis suppressor RKIP in downregulating signaling pathways involved with immune response in breast cancer cells and discuss its potential ramification on cancer therapy.

DHODH is an independent prognostic marker and potent therapeutic target in neuroblastoma.

Despite intensive therapy, children with high-risk neuroblastoma are at risk of treatment failure. We applied a multiomic system approach to evaluate metabolic vulnerabilities in human neuroblastoma. We combined metabolomics, CRISPR screening, and transcriptomic data across more than 700 solid tumor cell lines and identified dihydroorotate dehydrogenase (DHODH), a critical enzyme in pyrimidine synthesis, as a potential treatment target. Of note, DHODH inhibition is currently under clinical investigation in patients with hematologic malignancies. In neuroblastoma, DHODH expression was identified as an independent risk factor for aggressive disease, and high DHODH levels correlated to worse overall and event-free survival. A subset of tumors with the highest DHODH expression was associated with a dismal prognosis, with a 5-year survival of less than 10%. In xenograft and transgenic neuroblastoma mouse models treated with the DHODH inhibitor brequinar, tumor growth was dramatically reduced, and survival was extended. Furthermore, brequinar treatment was shown to reduce the expression of MYC targets in 3 neuroblastoma models in vivo. A combination of brequinar and temozolomide was curative in the majority of transgenic TH-MYCN neuroblastoma mice, indicating a highly active clinical combination therapy. Overall, DHODH inhibition combined with temozolomide has therapeutic potential in neuroblastoma, and we propose this combination for clinical testing.

Loss of negative feedback control of nuclear factor-kappaB2 activity in lymphocytes leads to fatal lung inflammation.

Proteolytic processing of the nuclear factor (NF)-kappaB2 precursor protein p100 generates the active NF-kappaB2 subunit p52, which in turn transcriptionally up-regulates p100 expression. p100 also functions as an IkappaB molecule capable of repressing p52 activity. The biological significance of this negative feedback control loop has yet to be demonstrated in vivo. Here we show that mice deficient in p100 but with constitutive expression of p52 in lymphocytes developed fatal lung inflammation characterized by diffuse alveolar damage with marked peribronchial fibrosis. In contrast, their littermates with only p100 deficiency or constitutive expression of p52 in lymphocytes developed mild lung inflammation with perivascular lymphocyte infiltration and had a normal life span. The fatal lung inflammation is associated with high-level induction of interferon-gamma and its inducible inflammatory chemokines, suggesting the involvement of a T-helper-1 immune response. These findings demonstrate the physiological relevance of the NF-kappaB2 p100 precursor protein in limiting the potentially detrimental effects of constitutive NF-kappaB2 signaling in lymphocytes.

NF-kappa B2 p100 is a pro-apoptotic protein with anti-oncogenic function.

Nuclear factor-kappa B (NF-kappa B) promotes cell survival by upregulating expression of anti-apoptotic genes, a process that is antagonized by inhibitors of kappa B (I kappa B) factors. The only NF-kappa B family member known to be mutated in human cancer is NF-kappa B2 p100 (ref. 2), a factor with I kappa B activity. Here, we report the isolation from irradiated mouse tumour cells of a complex that induces caspase-8 activity in cell-free assays and identify p100 as an essential component of this complex. Expression of p100 profoundly sensitizes cells to death-receptor-mediated apoptosis through a pathway that is independent of I kappa B-like activity. The carboxyl terminus of p100 contains a death domain that is absent from all known tumour-derived mutants. This death domain mediates recruitment of p100 into death machinery complexes after ligand stimulation and is essential for p100's pro-apoptotic activity. p100 also sensitizes NIH3T3 cells to apoptosis triggered by oncogenic Ras, resulting in a marked inhibition of transformation that is rescued by suppression of endogenous caspase-8. These observations thus identify an I kappa B-independent apoptotic activity of NF-kappa B2 p100 and help explain its unique tumour suppressor role.

Therapeutic targeting of both dihydroorotate dehydrogenase and nucleoside transport in MYCN-amplified neuroblastoma.

Metabolic reprogramming is an integral part of the growth-promoting program driven by the MYC family of oncogenes. However, this reprogramming also imposes metabolic dependencies that could be exploited therapeutically. Here we report that the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) is an attractive therapeutic target for MYCN-amplified neuroblastoma, a childhood cancer with poor prognosis. Gene expression profiling and metabolomic analysis reveal that MYCN promotes pyrimidine nucleotide production by transcriptional upregulation of DHODH and other enzymes of the pyrimidine-synthesis pathway. Genetic and pharmacological inhibition of DHODH suppresses the proliferation and tumorigenicity of MYCN-amplified neuroblastoma cell lines. Furthermore, we obtain evidence suggesting that serum uridine is a key factor in determining the efficacy of therapeutic agents that target DHODH. In the presence of physiological concentrations of uridine, neuroblastoma cell lines are highly resistant to DHODH inhibition. This uridine-dependent resistance to DHODH inhibitors can be abrogated by dipyridamole, an FDA-approved drug that blocks nucleoside transport. Importantly, dipyridamole synergizes with DHODH inhibition to suppress neuroblastoma growth in animal models. These findings suggest that a combination of targeting DHODH and nucleoside transport is a promising strategy to overcome intrinsic resistance to DHODH-based cancer therapeutics.

Using mammographic density to predict breast cancer risk: dense area or percentage dense area.

INTRODUCTION: Mammographic density (MD) is one of the strongest risk factors for breast cancer. It is not clear whether this association is best expressed in terms of absolute dense area or percentage dense area (PDA). METHODS: We measured MD, including nondense area (here a surrogate for weight), in the mediolateral oblique (MLO) mammogram using a computer-assisted thresholding technique for 634 cases and 1,880 age-matched controls from the Cambridge and Norwich Breast Screening programs. Conditional logistic regression was used to estimate the risk of breast cancer, and fits of the models were compared using likelihood ratio tests and the Bayesian information criteria (BIC). All P values were two-sided. RESULTS: Square-root dense area was the best single predictor (for example, χ1² = 53.2 versus 44.4 for PDA). Addition of PDA and/or square-root nondense area did not improve the fit (both P > 0.3). Addition of nondense area improved the fit of the model with PDA (χ1² = 11.6; P < 0.001). According to the BIC, the PDA and nondense area model did not provide a better fit than the dense area alone model. The fitted values of the two models were highly correlated (r = 0.97). When a measure of body size is included with PDA, the predicted risk is almost identical to that from fitting dense area alone. CONCLUSIONS: As a single parameter, dense area provides more information than PDA on breast cancer risk.

Predicting breast cancer risk using mammographic density measurements from both mammogram sides and views.

Mammographic density is a strong risk factor for breast cancer. Which and how many x-rays are used for research, and how mammographic density is measured varies across studies. In this article, we compared three different measurements (absolute dense area, percent dense area and percent dense volume) from each of four mammograms [left, right, medio-lateral oblique (MLO) and cranio-caudal (CC) views] using three different methods of measurement [computer-assisted thresholding, visual assessment and standard mammogram form (SMF)] to investigate whether additional measurements and/or different methods of measurement provide more information in the prediction of breast cancer risk. Mammographic density was measured in all four mammograms from 318 cases and 899 age-matched controls combined from the Cambridge and Norwich Breast Screening Programmes. Measurements were averaged across various combinations of mammogram type and/or view. Conditional logistic regression was used to estimate odds ratios associated with increasing quintiles of each mammographic measure. Overall, there appeared to be no difference in the fit of the models using two or four mammograms compared to the models using just the contralateral MLO or CC mammogram (all P > 0.07) for all methods of measurement. Common practice of measuring just the contralateral MLO or CC mammogram for analysis in case-control studies investigating the association between mammographic density and breast cancer risk appears to be sufficient.

MYCN promotes the expansion of Phox2B-positive neuronal progenitors to drive neuroblastoma development.

Amplification of the oncogene MYCN is a tumorigenic event in the development of a subset of neuroblastomas that commonly consist of undifferentiated or poorly differentiated neuroblasts with unfavorable clinical outcome. The cellular origin of these neuroblasts is unknown. Additionally, the cellular functions and target cells of MYCN in neuroblastoma development remain undefined. Here we examine the cell types that drive neuroblastoma development in TH-MYCN transgenic mice, an animal model of the human disease. Neuroblastoma development in these mice begins with hyperplastic lesions in early postnatal sympathetic ganglia. We show that both hyperplasia and primary tumors are composed predominantly of highly proliferative Phox2B(+) neuronal progenitors. MYCN induces the expansion of these progenitors by both promoting their proliferation and preventing their differentiation. We further identify a minor population of undifferentiated nestin(+) cells in both hyperplastic lesions and primary tumors that may serve as precursors of Phox2B(+) neuronal progenitors. These findings establish the identity of neuroblasts that characterize the tumor phenotype and suggest a cellular pathway by which MYCN can promote neuroblastoma development.

Mammographic density, estrogen receptor status and other breast cancer tumor characteristics.

The aim of this study was to examine the relationship between mammographic density and histological characteristics of breast tumors within a case-control study population. This study was an expansion of a large size case-control study examining the relationship between breast density and breast cancer risk. Percent and area of breast density was assessed in 370 invasive breast cancer cases and 1904 age-matched controls, using a computer-assisted method. Associations between breast density and estrogen receptor (ER) status, histological grade, histological size, lymph node status, vascular invasion, disease extent, and Nottingham Prognostic Index were evaluated, using logistic regression. Women with 50% or greater mammographic density have a 2.63-fold risk (95% confidence interval [95% CI] = 1.78-3.87; p < 0.001) of developing breast cancer compared to women with less than 10% density. Increase in every category of percentage of breast density is also associated with a 1.45-fold risk in developing ER positive tumors relative to ER negative tumors (odds ratio [OR] = 1.02; 95% CI = 1.00-1.04; p = 0.048), and increase in every quartile of absolute area of density is associated with a 1.48-fold ER positive breast cancer risk [95% CI = 1.06-2.07; p = 0.020]. Furthermore, breast density was found to be associated with specifically ER positivity, invasion as well as invasion with in situ, histological grades 1 and 2, tumor size larger than 1.1 cm, lack of vascular invasion, lymph node positivity and negativity, and NPI less than 4.0. After stratifying the data according to mode of diagnosis, the relationship became slightly stronger in the interval cancer group. Similar results were in observed using percent density and absolute density readings. Mammographic density was a stronger risk factor for ER positive [OR = 2.94; 95% CI = 1.94-4.43; p < 0.001] than ER negative cancers when comparing breasts with greater than 50% dense region to those with less than 10% density. No other tumor characteristic had a significant correlation with breast density. These results suggest that mammographic percent density may be more strongly related to ER positive than ER negative breast cancer, but otherwise is a risk factor for breast cancer independent of other tumor characteristics.

Histone demethylase KDM6B has an anti-tumorigenic function in neuroblastoma by promoting differentiation.

Induction of differentiation is a therapeutic strategy in high-risk neuroblastoma, a childhood cancer of the sympathetic nervous system. Neuroblastoma differentiation requires transcriptional upregulation of neuronal genes. How this process is regulated at epigenetic levels is not well understood. Here we report that the histone H3 lysine 27 demethylase KDM6B is an epigenetic activator of neuroblastoma cell differentiation. KDM6B mRNA expression is downregulated in poorly differentiated high-risk neuroblastomas and upregulated in differentiated tumors, and high KDM6B expression is prognostic for better survival in neuroblastoma patients. In neuroblastoma cell lines, KDM6B depletion promotes cell proliferation, whereas KDM6B overexpression induces neuronal differentiation and inhibits cell proliferation and tumorgenicity. Mechanistically, KDM6B epigenetically activates the transcription of neuronal genes by removing the repressive chromatin marker histone H3 lysine 27 trimethylation. In addition, we show that KDM6B functions downstream of the retinoic acid-HOXC9 axis in inducing neuroblastoma cell differentiation: KDM6B expression is upregulated by retinoic acid via HOXC9, and KDM6B is required for HOXC9-induced neuroblastoma cell differentiation. Finally, we present evidence that KDM6B interacts with HOXC9 to target neuronal genes for epigenetic activation. These findings identify a KDM6B-dependent epigenetic mechanism in the control of neuroblastoma cell differentiation, providing a rationale for reducing histone H3 lysine 27 trimethylation as a strategy for enhancing differentiation-based therapy in high-risk neuroblastoma.

Metabolic Reprogramming by MYCN Confers Dependence on the Serine-Glycine-One-Carbon Biosynthetic Pathway.

MYCN amplification drives the development of neuronal cancers in children and adults. Given the challenge in therapeutically targeting MYCN directly, we searched for MYCN-activated metabolic pathways as potential drug targets. Here we report that neuroblastoma cells with MYCN amplification show increased transcriptional activation of the serine-glycine-one-carbon (SGOC) biosynthetic pathway and an increased dependence on this pathway for supplying glucose-derived carbon for serine and glycine synthesis. Small molecule inhibitors that block this metabolic pathway exhibit selective cytotoxicity to MYCN-amplified cell lines and xenografts by inducing metabolic stress and autophagy. Transcriptional activation of the SGOC pathway in MYCN-amplified cells requires both MYCN and ATF4, which form a positive feedback loop, with MYCN activation of ATF4 mRNA expression and ATF4 stabilization of MYCN protein by antagonizing FBXW7-mediated MYCN ubiquitination. Collectively, these findings suggest a coupled relationship between metabolic reprogramming and increased sensitivity to metabolic stress, which could be exploited as a strategy for selective cancer therapy. SIGNIFICANCE: This study identifies a MYCN-dependent metabolic vulnerability and suggests a coupled relationship between metabolic reprogramming and increased sensitivity to metabolic stress, which could be exploited for cancer therapy.See related commentary by Rodriguez Garcia and Arsenian-Henriksson, p. 3818.

Evaluating the effectiveness of using standard mammogram form to predict breast cancer risk: case-control study.

Breast density is a well-known breast cancer risk factor. Most current methods of measuring breast density are area based and subjective. Standard mammogram form (SMF) is a computer program using a volumetric approach to estimate the percent density in the breast. The aim of this study is to evaluate the current implementation of SMF as a predictor of breast cancer risk by comparing it with other widely used density measurement methods. The case-control study comprised 634 cancers with 1,880 age-matched controls combined from the Cambridge and Norwich Breast Screening Programs. Data collection involved assessing the films based both on Wolfe's parenchymal patterns and on visual estimation of percent density and then digitizing the films for computer analysis (interactive threshold technique and SMF). Logistic regression was used to produce odds ratios associated with increasing categories of breast density. Density measures from all four methods were strongly associated with breast cancer risk in the overall population. The stepwise rises in risk associated with increasing density as measured by the threshold method were 1.37 [95% confidence interval (95% CI), 1.03-1.82], 1.80 (95% CI, 1.36-2.37), and 2.45 (95% CI, 1.86-3.23). For each increasing quartile of SMF density measures, the risks were 1.11 (95% CI, 0.85-1.46), 1.31 (95% CI, 1.00-1.71), and 1.92 (95% CI, 1.47-2.51). After the model was adjusted for SMF results, the threshold readings maintained the same strong stepwise increase in density-risk relationship. On the contrary, once the model was adjusted for threshold readings, SMF outcome was no longer related to cancer risk. The available implementation of SMF is not a better cancer risk predictor compared with the thresholding method.

KDM4C and ATF4 Cooperate in Transcriptional Control of Amino Acid Metabolism.

The histone lysine demethylase KDM4C is often overexpressed in cancers primarily through gene amplification. The molecular mechanisms of KDM4C action in tumorigenesis are not well defined. Here, we report that KDM4C transcriptionally activates amino acid biosynthesis and transport, leading to a significant increase in intracellular amino acid levels. Examination of the serine-glycine synthesis pathway reveals that KDM4C epigenetically activates the pathway genes under steady-state and serine deprivation conditions by removing the repressive histone modification H3 lysine 9 (H3K9) trimethylation. This action of KDM4C requires ATF4, a transcriptional master regulator of amino acid metabolism and stress responses. KDM4C activates ATF4 transcription and interacts with ATF4 to target serine pathway genes for transcriptional activation. We further present evidence for KDM4C in transcriptional coordination of amino acid metabolism and cell proliferation. These findings suggest a molecular mechanism linking KDM4C-mediated H3K9 demethylation and ATF4-mediated transactivation in reprogramming amino acid metabolism for cancer cell proliferation.