Twist2 amplification in rhabdomyosarcoma represses myogenesis and promotes oncogenesis by redirecting MyoD DNA binding.

Rhabdomyosarcoma (RMS) is an aggressive pediatric cancer composed of myoblast-like cells. Recently, we discovered a unique muscle progenitor marked by the expression of the Twist2 transcription factor. Genomic analyses of 258 RMS patient tumors uncovered prevalent copy number amplification events and increased expression of TWIST2 in fusion-negative RMS. Knockdown of TWIST2 in RMS cells results in up-regulation of MYOGENIN and a decrease in proliferation, implicating TWIST2 as an oncogene in RMS. Through an inducible Twist2 expression system, we identified Twist2 as a reversible inhibitor of myogenic differentiation with the remarkable ability to promote myotube dedifferentiation in vitro. Integrated analysis of genome-wide ChIP-seq and RNA-seq data revealed the first dynamic chromatin and transcriptional landscape of Twist2 binding during myogenic differentiation. During differentiation, Twist2 competes with MyoD at shared DNA motifs to direct global gene transcription and repression of the myogenic program. Additionally, Twist2 shapes the epigenetic landscape to drive chromatin opening at oncogenic loci and chromatin closing at myogenic loci. These epigenetic changes redirect MyoD binding from myogenic genes toward oncogenic, metabolic, and growth genes. Our study reveals the dynamic interplay between two opposing transcriptional regulators that control the fate of RMS and provides insight into the molecular etiology of this aggressive form of cancer.

NPAS2, transcriptionally activated by ARRB1, promotes the malignant behaviours of lung adenocarcinoma cells and regulates the reprogramming of glucose metabolism.

Lung adenocarcinoma (LUAD) is a serious threat to public health and is accompanied by increased morbidity and mortality worldwide. Neuronal PAS domain protein2 (NPAS2) has been confirmed as an oncogene in LUAD; however, little is known about its molecular mechanism. Here, the expression level of NPAS2 was detected in LUAD cell lines and 16HBE cells. Gain- and loss-of-function experiments were performed. Cell Counting Kit-8, colony formation, flow cytometry, wound-healing and Transwell assays were conducted to assess cell proliferation, apoptosis, migration and invasion, respectively. Reprogramming of glucose metabolism was evaluated via oxygen consumption rate (OCR), complexes activities, lactic production and glucose consumption. The expression of critical proteins was examined by western blot. We demonstrated aberrant upregulation of NPAS2 and ß-arrestin-1 (ARRB1) in LUAD cell lines. ARRB1 was found to be a critical transcription factor of NPAS2 with binding sites within the promoter region of NPAS2, thereby causing its transcriptional activation. Functional experiments revealed that NPAS2 depletion significantly inhibited the malignant behaviours of A549 cells by suppressing cell proliferation, migration, invasion and epithelial-mesenchymal transition and promoting cell apoptosis. Meanwhile, NPAS2 depletion increased OCR and activities of complexes (I, II, III and V), and reduced lactic acid production and glucose uptake in A549 cells, indicating that NPAS2 depletion inhibited aerobic glycolysis, accompanied by reduced expression of glycolytic enzymes. However, the changes caused by NPAS2 knockdown were partly restored by ARRB1 overexpression. In conclusion, our study suggests that ARRB1 could transcriptionally activate NPAS2, facilitating malignant activities and glycolysis, and ultimately promoting the progression of LUAD, proving a novel therapeutic strategy for the treatment of LUAD.

Temperature-compensated high strain measurement based on a Fabry-Perot interferometer with the virtual-assisted Vernier effect.

A highly sensitive optical fiber Fabry-Perot interferometer (FPI) for strain measurement with temperature compensation is proposed. Instead of using another actual reference interferometer, a virtual FPI is constructed to superpose with the sensing FPI to form the Vernier effect. The fundamental and the first-order harmonic Vernier effect are generated to increase the sensitivity by adjusting the parameter of the virtual FPI. In order to separate the strain from the environment temperature, an FBG is cascaded to distinguish the applied temperature. Experimental results demonstrate that, with the help of the fundamental Vernier effect, the sensitivity and temperature of the FPI increases from 1.05 pm/°C to 10.63 pm/°C in the temperature range of 40-120°C, and the sensitivity of strain increases from 2.635 pm/µÎµ to 33.11 pm/µÎµ in the strain range of 0-400 µÎµ. In order to access the tracking points more easily and further enhance the sensitivities, the first-order harmonic Vernier effect is generated by modifying the virtual FPI. Results show that the temperature and strain sensitivities are 21.25 pm/°C and 62.25 pm/µÎµ, respectively. In addition, with the help of the FBG, the strain can be separated from the temperature by solving the cross-sensitivity matrix.

Ectopic PLAG1 induces muscular dystrophy in the mouse.

Even though various genetic mutations have been identified in muscular dystrophies (MD), there is still a need to understand the biology of MD in the absence of known mutations. Here we reported a new mouse model of MD driven by ectopic expression of PLAG1. This gene encodes a developmentally regulated transcription factor known to be expressed in developing skeletal muscle, and implicated as an oncogene in certain cancers including rhabdomyosarcoma (RMS), an aggressive soft tissue sarcoma composed of myoblast-like cells. By breeding loxP-STOP-loxP-PLAG1 (LSL-PLAG1) mice into the MCK-Cre line, we achieved ectopic PLAG1 expression in cardiac and skeletal muscle. The Cre/PLAG1 mice died before 6 weeks of age with evidence of cardiomyopathy significantly limiting left ventricle fractional shortening. Histology of skeletal muscle revealed dystrophic features, including myofiber necrosis, fiber size variation, frequent centralized nuclei, fatty infiltration, and fibrosis, all of which mimic human MD pathology. QRT-PCR and Western blot revealed modestly decreased Dmd mRNA and dystrophin protein in the dystrophic muscle, and immunofluorescence staining showed decreased dystrophin along the cell membrane. Repression of Dmd by ectopic PLAG1 was confirmed in dystrophic skeletal muscle and various cell culture models. In vitro studies showed that excess IGF2 expression, a transcriptional target of PLAG1, phenocopied PLAG1-mediated down-regulation of dystrophin. In summary, we developed a new mouse model of a lethal MD due to ectopic expression of PLAG1 in heart and skeletal muscle. Our data support the potential contribution of excess IGF2 in this model. Further studying these mice may provide new insights into the pathogenesis of MD and perhaps lead to new treatment strategies.

Similarities and differences between internet gaming disorder and tobacco use disorder: A large-scale network study.

Studies have shown that internet gaming disorder (IGD) has the potential to be a type of addiction; however, direct comparisons (similarities and differences) between IGD and traditional addictions remain scarce, especially at the neuroimaging level. Resting-state functional magnetic resonance imaging (fMRI) data were collected from 92 individuals with IGD, 96 individuals with tobacco use disorders (TUDs) and 107 individuals who served as healthy controls (HCs). Independent component analysis (ICA) was performed to explore the similarities and differences among these three groups; Granger causality analysis (GCA) was further performed based on the ICA results to determine potential neural features underlying the differences and similarities among the groups. The ICA results indicated significant differences in the subcortical network and cerebellar network. GCA results found that significant differences in bilateral caudate among three groups, and the efferents of dorsal frontostriatal circuit showed significant differences in insula among three groups, whereas efferents of ventral frontostriatal circuit showed significant differences in the medial prefrontal cortex (mPFC). Two kinds of addiction showed differences in thalamus and frontostriatal circuits, and similar changes found in cerebellum and mPFC regions. It suggested that addiction disorders have psychopathology features, and the craving and reward dysfunctions may be the key reasons. Although both substance addiction and behaviour addiction showed craving dysfunction in cerebellum, however, the key reward dysfunction of substance addiction was found in subcortical regions, whereas behaviour addiction located in cortical regions.

A distant, cis-acting enhancer drives induction of Arf by Tgfß in the developing eye.

The Arf tumor suppressor represents one of several genes encoded at the Cdkn2a and Cdkn2b loci in the mouse. Beyond its role blunting the growth of incipient cancer cells, the Arf gene also plays an essential role in development: its gene product, p19(Arf), is induced by Tgfß2 in the developing eye to dampen proliferative signals from Pdgfrß, which effect ultimately fosters the vascular remodeling required for normal vision in the mouse. Mechanisms underlying Arf induction by Tgfß2 are not fully understood. Using the chr4(Δ70 kb/Δ70 kb) mouse, we now show that deletion of the coronary artery disease (CAD) risk interval lying upstream of the Cdkn2a/b locus represses developmentally-timed induction of Arf resulting in eye disease mimicking the persistent hyperplastic primary vitreous (PHPV) found in Arf-null mice and in children. Using mouse embryo fibroblasts, we demonstrate that Arf induction by Tgfß is blocked in cis to the 70 kb deletion, but Arf induction by activated RAS and cell culture "shock" is not. Finally, we show that Arf induction by Tgfß is derailed by preventing RNA polymerase II recruitment following Smad 2/3 binding to the promoter. These findings provide the first evidence that the CAD risk interval, located at a distance from Arf, acts as a cis enhancer of Tgfß2-driven induction of Arf during development.

Wanting-liking dissociation and altered dopaminergic functioning: Similarities between internet gaming disorder and tobacco use disorder.

Background: Although internet gaming disorder (IGD) has been included in the DSM-5 for approximately 10 years, debate remains regarding its existence and classification. Methods: The current research incorporated three approaches. First, implicit association tests were used to examine for potential dissociation between wanting and liking in IGD. Second, brain features in wanting and liking circuits were tested and compared with tobacco use disorder (TUD) when performing a cue-craving task to explore the neural features of wanting and liking. Third, dopaminergic systems were investigated in IGD and TUD using neuromelanin-sensitive MRI. Results: The implicit association test results supported a wanting-liking dissociation in IGD participants. Functional MRI data suggested neural correlates underlying wanting-liking dissociation in IGD and TUD participants, with positive correlations suggesting greater dissociation with increasing addiction severity. Neuromelanin results suggest dopaminergic differences in IGD and TUD relative to healthy control participants. Conclusions: A wanting-liking dissociation in IGD participants suggests gaming motivations in IGD relating to incentive sensitization rather than hedonic responses. The neuromelanin-sensitive MRI results suggest dopaminergic involvement in IGD and TUD. The findings suggest similar brain-behaviour mechanisms for IGD and TUD based on an incentive-sensitization model for addiction, having implications for potential therapeutic strategies and policy-based interventions.

rTMS effects on urges and severity of tobacco use disorder operate independently of a retrieval-extinction component and involve frontal-striatal pathways.

BACKGROUND: Although smoking remains a leading cause of preventable disease, the treatment options for smoking are limited. The present study evaluated the neural features underlying effects of repetitive transcranial magnetic stimulation (rTMS) for reducing smoking cravings. In addition, the efficacy of a simulated retrieval-extinction procedure to augment rTMS efficacy was examined. METHODS: Sixty-one individuals with tobacco use disorder (TUD) were randomized into three groups: classic rTMS, retrieval rTMS (viewed smoking videos before rTMS), and sham rTMS. rTMS was performed on the left dorsolateral prefrontal cortex (DLPFC) over 5 days using a standard figure-8 coil. Smoking cravings and brain responses to smoking cues were measured before and after rTMS treatment. Changes in functional connectivity (FC) among different brain regions were calculated. RESULTS: rTMS reduced smoking urges in TUD. Both active-rTMS groups demonstrated greater activations of the DLPFC, caudate, and bilateral insula relative to the sham group. Increased FC was observed between executive and reward network brain regions, and decreased FC was observed within reward network regions. Compared with standard rTMS, retrieval-extinction rTMS demonstrated similar outcomes and was associated with less activation of the medial frontal gyrus. CONCLUSIONS: rTMS increased activations in brain regions implicated in executive control and reward processing. Strengthened prefrontal-striatal pathway suggests that rTMS enhanced top-down control over smoking cravings. The retrieval-extinction process, although associated with some different and multiple similar neural correlates as the standard rTMS, did not enhance cessation outcomes.

A20 affects macrophage polarization through the NLRP3 inflammasome signaling pathway and promotes breast cancer progression.

Breast cancer is the most common malignant tumor in females, and the majority of patients succumb to metastasis. The present study aimed to investigate the association between tumor necrosis factor alpha-induced protein 3 (A20), NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) and tumor-associated macrophage polarization, and their effects on the proliferation and metastasis of breast cancer cells. The expression of A20 in breast cancer cells was analyzed by reverse transcription-quantitative PCR (RT-qPCR) and western blotting. RT-qPCR and western blotting were also used to confirm the transfection efficiency. The viability, clone formation, migration, invasion and angiogenesis of transfected breast cancer cells were detected by Cell Counting Kit-8, colony formation, wound healing, Transwell and tube formation assays, respectively. Activated macrophages, namely M1 and M2 type macrophages, were observed by double staining immunofluorescence. The levels of M1 and M2 macrophage markers were analyzed by qPCR. The expression of angiogenesis-related proteins and NLRP3 inflammasome activation-associated proteins was detected by western blotting. The results revealed that A20 was highly expressed in breast cancer cells. Interference with A20 inhibited the proliferation, invasion, migration and angiogenesis of breast cancer cells, and inhibited the M2-like polarization of macrophages. Interference with A20 promoted the activation of the NLRP3 inflammasome. The NLRP3 inhibitor MCC950 alleviated the effect of interference with A20 to promote macrophage proliferation and recruitment, as well as M2-like polarization. In conclusion, interference with A20 inhibited macrophage proliferation and M2-like polarization through the NLRP3 inflammasome signaling pathway to inhibit breast cancer progression.

Resting state default mode network is associated with wise advising.

Default mode network (DMN) may be associated with wisdom (i.e., mature understanding of life featured by perspectival metacognition) when advising from a self-referential perspective due to the involvement of the DMN in reflecting on personal life experiences. After a resting-state functional MRI scan, 52 adults advised some youths going through life dilemmas, half from a second-person perspective and half from a third. After advising each youth, participants indicated the psychological distance they felt between themselves and the youth. The amplitude of low-frequency fluctuation (ALFF) was measured in the DMN during resting states. Moreover, trained raters rated the participants' advice on wisdom criteria (i.e., metacognitive humility (MH), meta-level flexibility, and perspective-taking). The results showed that participants felt a significantly smaller psychological distance from the youth when advising from the second- than the third-person perspective. Moreover, only when advising from the second-person perspective was MH associated with ALFF in regions within the DMN (i.e., right rostral anterior cingulate cortex (ACC) and left dorsomedial prefrontal cortex). The right rostral ACC showed a significantly greater association with MH from the second- than the third-person perspective. Therefore, resting-state DMN activities may be important for self-involved wisdom performance (e.g., giving advice directly to others).

How sleep disturbances affect internet gaming disorder: The mediating effect of hippocampal functional connectivity.

BACKGROUND: Studies have revealed that sleep disturbances lead to an increased risk of Internet gaming disorder (IGD). However, the neural underpinnings of this feature remain unknown. Exploring this issue would be valuable in understanding the relationship between sleep and psychiatric disorders. METHODS: Given the impact of sleep on reward circuitry, we examined nucleus accumbens (NAcc) and hippocampal resting-state functional connectivity (rsFC) differences between 41 IGD subjects and 59 healthy controls. Significant connections were determined and used to examine correlations with clinical variables. Finally, we explored the relationship between neuroimaging findings, IGD severity and sleep disturbances through a mediation model. RESULTS: We observed the connection deviation between the hippocampus and a wide range of cerebral cortexes in IGD subjects, including the prefrontal, parietal and temporal lobes. More importantly, the right posterior hippocampus (pHIP)-left caudate rsFC was positively correlated with both the Pittsburgh Sleep Quality Index (PSQI) and Internet Addiction Test scores and mediated the relationship between the two. For the NAcc, a difference between groups was only observed in the rsFC between the shell partition of the NAcc and the inferior orbitofrontal cortex, but this connectivity was not related to the PSQI score. CONCLUSIONS: IGD subjects showed a wide range of abnormal connections in the hippocampus, involving memory, reward motivation, and cognitive control. Here we emphasized the potential of the hippocampus in studying sleep disturbances in IGD, especially the coupling between the pHIP and caudate nucleus, which could provide novel insight into how sleep interacts with motivational systems in IGD subjects.

Exon skipping in genes encoding lineage-defining myogenic transcription factors in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a childhood sarcoma composed of myoblast-like cells, which suggests a defect in terminal skeletal muscle differentiation. To explore potential defects in the differentiation program, we searched for mRNA splicing variants in genes encoding transcription factors driving skeletal muscle lineage commitment and differentiation. We studied two RMS cases and identified altered splicing resulting in "skipping" the second of three exons in MYOD1. RNA-Seq data from 42 tumors and additional RMS cell lines revealed exon 2 skipping in both MYOD1 and MYF5 but not in MYF6 or MYOG. Complementary molecular analysis of MYOD1 mRNA found evidence for exon skipping in 5 additional RMS cases. Functional studies showed that so-called MYODΔEx2 protein failed to robustly induce muscle-specific genes, and its ectopic expression conferred a selective advantage in cultured fibroblasts and an RMS xenograft. In summary, we present previously unrecognized exon skipping within MYOD1 and MYF5 in RMS, and we propose that alternative splicing can represent a mechanism to alter the function of these two transcription factors in RMS.

Tgfbeta signaling directly induces Arf promoter remodeling by a mechanism involving Smads 2/3 and p38 MAPK.

We have investigated how the Arf gene product, p19(Arf), is activated by Tgfß during mouse embryo development to better understand how this important tumor suppressor is controlled. Taking advantage of new mouse models, we provide genetic evidence that Arf lies downstream of Tgfß signaling in cells arising from the Wnt1-expressing neural crest and that the anti-proliferative effects of Tgfß depend on Arf in vivo. Tgfß1, -2, and -3 (but not BMP-2, another member of the Tgfß superfamily) induce p19(Arf) expression in wild type mouse embryo fibroblasts (MEFs), and they enhance Arf promoter activity in Arf(lacZ/lacZ) MEFs. Application of chemical inhibitors of Smad-dependent and -independent pathways show that SB431542, a Tgfß type I receptor (TßrI) inhibitor, and SB203580, a p38 MAPK inhibitor, impede Tgfß2 induction of Arf. Genetic studies confirm the findings; transient knockdown of Smad2, Smad3, or p38 MAPK blunt Tgfß2 effects, as does Cre recombinase treatment of Tgfbr2(fl/fl) MEFs to delete Tgfß receptor II. Chromatin immunoprecipitation reveals that Tgfß rapidly induces Smads 2/3 binding and histone H3 acetylation at genomic DNA proximal to Arf exon 1ß. This is followed by increased RNA polymerase II binding and progressively increased Arf primary and mature transcripts from 24 through 72 h, indicating that increased transcription contributes to p19(Arf) increase. Last, Arf induction by oncogenic Ras depends on p38 MAPK but is independent of TßrI activation of Smad 2. These findings add to our understanding of how developmental and tumorigenic signals control Arf expression in vivo and in cultured MEFs.

The imbalance between goal-directed and habitual systems in internet gaming disorder: Results from the disturbed thalamocortical communications.

BACKGROUND: Converging evidence has identified the imbalance between goal-directed systems and habitual systems in the addiction process. The thalamocortical loop plays an important role in the habitual/goal-directed system. However, little is known about the role of the thalamus in goal-directed and habitual systems in Internet gaming disorder (IGD) patients. This study investigated whether thalamocortical circuit was disrupted and how they affected goal-directed and habitual behaviors in IGD patients. METHODS: This is a functional magnetic resonance imaging (fMRI) study. Twenty-five IGD patients and 25 matched recreational game users (RGUs) were scanned when they were in a resting state and were performing an instrumental learning task to obtain behavioral data related to habitual/goal-directed behavior. We used the whole-brain seed-based functional connectivity (FC) of the four thalamic nuclei (bilateral) and correlation analyses to examine the thalamocortical loop difference and relationship with habitual/goal-directed performance. RESULTS: Compared with RGUs, IGD patients demonstrated significantly increased FC between the left midline nucleus (MN) and the right postcentral gyrus (PCG), and between the pulvinar and medial frontal gyrus (MFG). Correlation results showed that within the IGD group, the correct response rates of the participants to inconsistent stimulus-result pairs were positively correlated with the FC between the pulvinar and MFG. Inhibition-control scores were negatively correlated with the FC between the left MN and the PCG. CONCLUSIONS: IGD patients showed disrupted thalamocortical communication that could further result in an imbalance between the goal-directed and habitual systems in IGD patients. These findings provide more information about the involvement of the thalamus in the pathophysiology of IGD, and as potential circuit-level biomarkers of IGD patients, these circuit alterations may be useful in treatment development and in monitoring treatment outcomes.

[Analysis of the Predictive Value of EZH2 Expression Level on the Clinical Efficacy and Long-term Prognosis of Patients with Primary Gastrointestinal Diffuse Large B-cell Lymphoma].

OBJECTIVE: To investigate the predictive value of methyltransferase EZH2 expression level on the clinical efficacy and long-term prognosis of patients with primary gastrointestinal diffuse large B-cell lymphoma (PGI-DLBCL). METHODS: 161 patients with newly treated PGI-DLBCL in our hospital from August 2013 to July 2019 were selected. The expression level of EZH2 protein was detected by immunohistochemistry, and the short-term efficacy and long-term survival differences of patients with different levels of EZH2 were compared. The predictive values of EZH2 expression level on the short-term efficacy and long-term prognosis of PGI-DLBCL patients were analyzed by Log-rank test and COX risk proportional regression model. Chi-square test and Logistic regression analysis were used to analyze the influencing factors of EZH2 expression level. RESULTS: The complete response (CR) and overal response(OR) rates of those with high EZH2 expression were significantly lower than those with low EZH2 expression (P<0.001). The median OS and PFS of EZH2 high-level and low-level expression group was 37, 31 months and 49, 42 months, respectively. The cumulative OS and PFS rates of the high-level expression group were significantly lower than those of the low-level expression group, and the differences were statistically significant (P<0.05). The high expression levels of H3K27me3, EZH2, BCL-2, BCL-6, c-MYC were closely related to the shortening of OS and PFS, while the high expression level of Ki-67 was closely related to the shortening of OS (P<0.05), of which the high expression levels of H3K27me3, EZH2, BCL-2, and BCL-6 were independent risk factors for shortening of OS and PFS. The expression level of EZH2 was positively correlated with the expression level of H3K27me3, BCL-6, c-MYC and Ki-67 (r=0.741, r=0.837, r=0.809, r=0.772), and the high expression levels of H3K27me3, BCL-6 and Ki-67 were independent factors influencing the high expression of EZH2. CONCLUSION: In patients with PGI-DLBCL, the high expression of EZH2 significantly reduces the short-term CR and OR rates, which is an independent risk factor for the shortening of long-term OS and PFS rates, and it is independently related to the high expression of H3K27me3 and BCL6.

Latent Epstein-Barr virus infection status and prognosis in patients with newly diagnosed Hodgkin lymphoma in Southeast China: a single-center retrospective study.

PURPOSE: This study aimed to investigate the latent Epstein-Barr virus (EBV) infection status of patients with newly diagnosed Hodgkin lymphoma (HL) and to discuss the relationship between tumor cell EBV status and the prognosis of HL patients. PATIENTS AND METHODS: A total of 134 previously untreated HL patients were analyzed in the study. Epstein-Barr virus encoded RNAs (EBERs) in situ hybridization was performed to detect the EBV status of tumor cells. RESULTS: EBV positive status correlated with sex (p=0.046) and the proportion of extranodal lesions(p=0.037). There was no obvious correlation between EBV status and overall survival (OS) or failure-free survival (FFS) in all cases, but in cases over 50 years old, EBV positive group had an inferior 5-year FFS compared with EBV negative group (38.5%±13.5% vs 90.9%±8.7%, p=0.012). In FFS multivariate analysis of this age subgroup, EBV positive status was associated with significantly inferior survival (HR, 10.10; 95% CI, 1.26-81.08; p=0.030). CONCLUSION: This study demonstrates positive tumor cell EBV status is an unfavorable prognostic factor in elder HL patients.

More stringent criteria are needed for diagnosing internet gaming disorder: Evidence from regional brain features and whole-brain functional connectivity multivariate pattern analyses.

BACKGROUND: Internet gaming disorder (IGD) is included in the DSM-5 as a provisional diagnosis. Whether IGD should be regarded as a disorder and, if so, how it should be defined and thresholded have generated considerable debate. METHODS: In the current study, machine learning was used, based on regional and interregional brain features. Resting-state data from 374 subjects (including 148 IGD subjects with DSM-5 scores ≥5 and 93 IGD subjects with DSM-5 scores ≥6) were collected, and multivariate pattern analysis (MVPA) was employed to classify IGD from recreational game use (RGU) subjects based on regional brain features (ReHo) and communication between brain regions (functional connectivity; FC). Permutation tests were used to assess classifier performance. RESULTS: The results demonstrated that when using DSM-5 scores ≥5 as the inclusion criteria for IGD subjects, MVPA could not differentiate IGD subjects from RGU, whether based on ReHo or FC features or by using different templates. MVPA could differentiate IGD subjects from RGU better than expected by chance when using DSM-5 scores ≥6 with both ReHo and FC features. The brain regions involved in the default mode network and executive control network and the cerebellum exhibited high discriminative power during classification. DISCUSSION: The current findings challenge the current IGD diagnostic criteria thresholding proposed in the DSM-5, suggesting that more stringent criteria may be needed for diagnosing IGD. The findings suggest that brain regions involved in the default mode network and executive control network relate importantly to the core criteria for IGD.

Bayesian Modeling Identifies PLAG1 as a Key Regulator of Proliferation and Survival in Rhabdomyosarcoma Cells.

We recently developed a novel computational algorithm that incorporates Bayesian methodology to identify rhabdomyosarcoma disease genes whose expression level correlates with copy-number variations, and we identified PLAG1 as a candidate oncogenic driver. Although PLAG1 has been shown to contribute to other type of cancers, its role in rhabdomyosarcoma has not been elucidated. We observed that PLAG1 mRNA is highly expressed in rhabdomyosarcoma and is associated with PLAG1 gene copy-number gain. Knockdown of PLAG1 dramatically decreased cell accumulation and induced apoptosis in rhabdomyosarcoma cells, whereas its ectopic expression increased cell accumulation in vitro and as a xenograft and promoted G1 to S-phase cell-cycle progression. We found that PLAG1 regulates IGF2 expression and influences AKT and MAPK pathways in rhabdomyosarcoma, and IGF2 partially rescues cell death triggered by PLAG1 knockdown. The expression level of PLAG1 correlated with the IC50 of rhabdomyosarcoma cells to BMS754807, an IGF receptor inhibitor. IMPLICATIONS: Our data demonstrate that PLAG1 contributes to proliferation and survival of rhabdomyosarcoma cells at least partially by inducing IGF2, and this new understanding may have the potential for clinical translation.

Identification of De Novo Enhancers Activated by TGFß to Drive Expression of CDKN2A and B in HeLa Cells.

Disruption of the CDKN2A (INK4A/ARF) and B (INK4B) genes, which encode three function-independent tumor suppressors, is one of the most common events in human cancer. Because their relative importance in tumor prevention appears to be species- and context-specific, studying their regulation can shed light on mechanisms by which they are bypassed in malignant transformation. We previously unveiled a new pathway in which TGFß selectively induces Arf at mouse Cdkn2a in eye development and cultured fibroblasts. As TGFß signaling is often derailed in cancer development or progression, we investigated its control of CDKN2A/B in human cancer. Computational analyses of sequencing and array data from nearly 11,000 patients with cancer in TCGA showed discordant expression of ARF and INK4A in most cancer subtypes, with gene copy-number loss and promoter methylation involved in only a subset. Using HeLa cells as a model, we found that exogenous TGFß induced ARF mRNA and protein, and ARF knockdown limited TGFß-mediated growth suppression. TGFß-mediated ARF mRNA induction required SMAD2/3, p38MAPK, and SP1, and ARF mRNA was induced without added RNAPII recruitment. Chromatin immunoprecipitation unveiled a remote enhancer element engaged by TGFß by a mechanism that partially depended on p38MAPK. CRISPR-based editing of this enhancer limited induction of ARF and INK4B by TGFß, but not by oncogenic RAS. IMPLICATIONS: Our findings reveal new molecular mechanisms by which CDKN2A/B regulation is coupled to external cues, and those findings represent entry points to further explore pharmacologic strategies to restore their expression in cancer.

Novel PDGFRB rearrangement in multifocal infantile myofibromatosis is tumorigenic and sensitive to imatinib.

Infantile myofibromatosis (IM) is an aggressive neoplasm composed of myofibroblast-like cells in children. Although typically localized, it can also present as multifocal disease, which represents a challenge for effective treatment. IM has previously been linked to activating somatic and germline point mutations in the PDGFRß tyrosine kinase encoded by the PDGFRB gene. Clinical panel-based targeted tumor sequencing of a tumor from a newborn with multifocal IM revealed a novel PDGFRB rearrangement, which was reported as being of unclear significance. Additional sequencing of cDNA from tumor and germline DNA confirmed a complex somatic/mosaic PDGFRB rearrangement with an apparent partial tandem duplication disrupting the juxtamembrane domain. Ectopic expression of cDNA encoding the mutant form of PDGFRB markedly enhanced cell proliferation of mouse embryo fibroblasts (MEFs) compared to wild-type PDGFRB and conferred tumor-forming capacity on nontumorigenic 10T1/2 fibroblasts. The mutated protein enhanced MAPK activation and retained sensitivity to the PDGFRß inhibitor imatinib. Our findings reveal a new mechanism by which PDGFRB can be activated in IM, suggest that therapy with tyrosine kinase inhibitors including imatinib may be beneficial, and raise the possibility that this receptor tyrosine kinase might be altered in a similar fashion in additional cases that would similarly present annotation challenges in clinical DNA sequencing analysis pipelines.