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Post-zygotically acquired genetic variants, or somatic variants, that arise during cortical development have emerged as important causes of focal epilepsies, particularly those due to malformations of cortical development. Pathogenic somatic variants have been identified in many genes within the PI3K-AKT-mTOR-signalling pathway in individuals with hemimegalencephaly and focal cortical dysplasia (type II), and more recently in SLC35A2 in individuals with focal cortical dysplasia (type I) or non-dysplastic epileptic cortex. Given the expanding role of somatic variants across different brain malformations, we sought to delineate the landscape of somatic variants in a large cohort of patients who underwent epilepsy surgery with hemimegalencephaly or focal cortical dysplasia. We evaluated samples from 123 children with hemimegalencephaly (n = 16), focal cortical dysplasia type I and related phenotypes (n = 48), focal cortical dysplasia type II (n = 44), or focal cortical dysplasia type III (n = 15). We performed high-depth exome sequencing in brain tissue-derived DNA from each case and identified somatic single nucleotide, indel and large copy number variants. In 75% of individuals with hemimegalencephaly and 29% with focal cortical dysplasia type II, we identified pathogenic variants in PI3K-AKT-mTOR pathway genes. Four of 48 cases with focal cortical dysplasia type I (8%) had a likely pathogenic variant in SLC35A2. While no other gene had multiple disease-causing somatic variants across the focal cortical dysplasia type I cohort, four individuals in this group had a single pathogenic or likely pathogenic somatic variant in CASK, KRAS, NF1 and NIPBL, genes previously associated with neurodevelopmental disorders. No rare pathogenic or likely pathogenic somatic variants in any neurological disease genes like those identified in the focal cortical dysplasia type I cohort were found in 63 neurologically normal controls (P = 0.017), suggesting a role for these novel variants. We also identified a somatic loss-of-function variant in the known epilepsy gene, PCDH19, present in a small number of alleles in the dysplastic tissue from a female patient with focal cortical dysplasia IIIa with hippocampal sclerosis. In contrast to focal cortical dysplasia type II, neither focal cortical dysplasia type I nor III had somatic variants in genes that converge on a unifying biological pathway, suggesting greater genetic heterogeneity compared to type II. Importantly, we demonstrate that focal cortical dysplasia types I, II and III are associated with somatic gene variants across a broad range of genes, many associated with epilepsy in clinical syndromes caused by germline variants, as well as including some not previously associated with radiographically evident cortical brain malformations.

ImportanceMesial temporal lobe epilepsy (MTLE) is the most common focal epilepsy subtype and is often refractory to anti-seizure medications. While most MTLE patients do not have pathogenic germline genetic variants, the contribution of post-zygotic (i.e., somatic) variants in the brain is unknown.ObjectiveTo test the association between pathogenic somatic variants in the hippocampus and MTLE.DesignThis case-control genetic association study analyzed the DNA derived from hippocampal tissue of neurosurgically-treated patients with MTLE and age- and sex-matched neurotypical controls. Participants were enrolled from 1988 through 2019 and clinical data was collected retrospectively. Whole-exome and gene-panel sequencing (depth>500X) were used to identify candidate pathogenic somatic variants. A subset of novel variants were functionally evaluated using cellular and molecular assays.SettingLevel 4 epilepsy centers, multi-center study.ParticipantsNon-lesional and lesional (mesial temporal sclerosis, focal cortical dysplasia, and low-grade epilepsy-associated tumors) drug-resistant MTLE patients who underwent anterior medial temporal lobectomy. All patients with available frozen tissue and appropriate consents were included. Control brain tissue was obtained from neurotypical donors at brain banks.ExposuresDrug-resistant MTLE.Main Outcomes and MeasuresPresence and abundance of pathogenic somatic variants in the hippocampus versus the unaffected temporal neocortex.ResultsSamples were obtained from 105 MTLE patients (52 male, 53 female; age: MED [IQR], 32 [26-44]) and 30 neurotypical controls (19 male, 11 female; age: MED [IQR], 37 [18-53]). Eleven pathogenic somatic variants, enriched in the hippocampus relative to the unaffected temporal neocortex (MED [IQR], 1.92 [1.5-2.7] vs 0.3 [0-0.9], pPTPN11, SOS1, KRAS, BRAF, andNF1, all predicted to constitutively activate Ras/Raf/MAPK signaling. Immunohistochemical studies of variant-positive hippocampal tissue demonstrated increased Erk1/2 phosphorylation, indicative of Ras/Raf/MAPK activation, predominantly in glial cells. Molecular assays showed abnormal liquid-liquid phase separation for thePTPN11variants as a possible dominant gain-of-function mechanism.Conclusions and RelevanceHippocampal somatic variants, particularly those activating Ras/Raf/MAPK signaling, may contribute to the pathogenesis of sporadic, drug-resistant MTLE. These findings may provide a novel genetic mechanism and highlight new therapeutic targets for this common indication for epilepsy surgery.

Post-zygotically acquired genetic variants, or somatic variants, that arise during cortical development have emerged as important causes of focal epilepsies, particularly those due to malformations of cortical development. Pathogenic somatic variants have been identified in many genes within the PI3K-AKT3-mTOR-signaling pathway in individuals with hemimegalencephaly and focal cortical dysplasia (type II), and more recently in SLC35A2 in individuals with focal cortical dysplasia (type I) or non-dysplastic epileptic cortex. Given the expanding role of somatic variants across different brain malformations, we sought to delineate the landscape of somatic variants in a large cohort of patients who underwent epilepsy surgery with hemimegalencephaly or focal cortical dysplasia. We evaluated samples from 123 children with hemimegalencephaly (n=16), focal cortical dysplasia type I and related phenotypes (n=48), focal cortical dysplasia type II (n=44), or focal cortical dysplasia type III (n=15) classified using imaging and pathological findings. We performed high-depth exome sequencing in brain tissue-derived DNA from each case and identified somatic single nucleotide, indel, and large copy number variants. In 75% of individuals with hemimegalencephaly and 29% with focal cortical dysplasia type II, we identified pathogenic variants in PI3K-AKT-mTOR pathway genes. Four of 48 cases with focal cortical dysplasia type I (8%) had a likely pathogenic variant in SLC35A2. While no other gene had multiple disease-causing somatic variants across the focal cortical dysplasia type I cohort, four individuals in this group had a single pathogenic or likely pathogenic somatic variant in CASK, KRAS, NF1, and NIPBL, genes associated with neurodevelopmental disorders. No rare pathogenic or likely pathogenic somatic variants in any neurological disease genes like those identified in the focal cortical dysplasia type I cohort were found in 63 neurologically normal controls (P = 0.017), suggesting a role for these novel variants. We also identified a somatic loss-of-function variant in the known epilepsy gene, PCDH19, present in a very small number of alleles in the dysplastic tissue from a female patient with focal cortical dysplasia IIIa with hippocampal sclerosis. In contrast to focal cortical dysplasia type II, neither focal cortical dysplasia type I nor III had somatic variants in genes that converge on a unifying biological pathway, suggesting greater genetic heterogeneity compared to type II. Importantly, we demonstrate that FCD types I, II, and III, are associated with somatic gene variants across a broad range of genes, many associated with epilepsy in clinical syndromes caused by germline variants, as well as including some not previously associated with radiographically evident cortical brain malformations.

WW domain-containing transcriptional regulator 1 (TAZ) is a transcriptional co-activator and effector of the Hippo signaling pathway. In certain breast cancer subtypes, Hippo signaling is dysregulated leading to activation of TAZ and altered expression of TAZ transcriptional targets. Over the past decade, we and others have found that TAZ transcriptionally regulates genes that affect multiple aspects of breast cancer cell behaviour. However, while cancer cell-intrinsic oncogenic functions of TAZ have emerged, less is known about whether TAZ might also contribute to tumourigenesis by sensitizing tumour cells to factors present in the tumour microenvironment or in systemic circulation. Here, we show that TAZ directly regulates the expression of insulin receptor substrate 1 (IRS1) in breast cancer cells. TAZ or IRS1 overexpression induces a similar proliferative transformation phenotype in MCF10A mammary epithelial cells. TAZ enhances IRS1 mRNA, protein levels and downstream signaling in MCF10A. Mechanistically, TAZ interacts with the IRS1 promoter through the TEAD family of transcription factors and enhances its activity. Critically, TAZ-induced IRS1 upregulation contributes to the proliferation of TAZ-overexpressing MCF10A in 3-dimensional (3D) Matrigel culture. Therefore, we offer compelling evidence that TAZ regulates signaling through the insulin pathway in breast cancer cells. These findings highlight an additional mechanism by which TAZ may promote breast cancer tumourigenesis and progression by modulating cancer cell responses to exogenously produced factors.

Purpose The availability of take home naloxone (THN) was increased for Canadians in 2016, including access to kits via pharmacies. Unlike typical over-the-counter (OTC) and prescription drugs, THN kits may be stored in non-standard conditions, including in vehicles, backpacks, and out of doors. To evaluate whether these non-standard storage conditions affect stability, we investigated the impact of heat and freeze-thaw cycling on naloxone hydrochloride stability. Methods To assess the effect of heat, naloxone hydrochloride ampoules were exposed to 80 °C in a temperature-controlled oven for 8 h followed by 16 h at room temperature. To assess the effect of freeze-thaw cycles, naloxone hydrochloride ampoules were exposed to − 20 °C for 16 h followed by 8 h at 4 °C. The impact of these conditions on naloxone hydrochloride stability was evaluated each day for 1 week and after 2 and 4 weeks. The concentration of remaining naloxone hydrochloride was quantified using high-performance liquid chromatography (HPLC). Naloxone hydrochloride ampoules stored at room temperature served as the experimental control. Results Naloxone hydrochloride ampoules exhibit no changes in drug concentration following exposure to heat or freeze-thaw cycles for up to 28 days compared to ampoules maintained at room temperature (as indicated in the product monograph). Conclusions Naloxone hydrochloride remains chemically stable following exposure to heat or freeze-thaw cycles after 28 days. If THN kits are stored in non-standard conditions (for up to 28 days) the active naloxone is likely to remain stable. Despite this, pharmacists should continue to emphasize the importance of appropriate storage of THN kits to ensure optimal efficacy should naloxone administration be required in an emergency situation. Electronic supplementary material The online version of this article (10.1186/s12954-019-0288-4) contains supplementary material, which is available to authorized users.

Transcriptional co-activator with a PDZ binding domain (TAZ) is a WW domain-containing transcriptional co-activator and a core component of an emerging Hippo signaling pathway that regulates organ size, tumorigenesis, metastasis, and drug resistance. TAZ regulates these biological functions by up-regulating downstream cellular genes through transactivation of transcription factors such as TEAD and TTF1. To understand the molecular mechanisms underlying TAZ-induced tumorigenesis, we have recently performed a gene expression profile analysis by overexpressing TAZ in mammary cells. In addition to the TAZ-up-regulated genes that were confirmed in our previous studies, we identified a large number of cellular genes that were down-regulated by TAZ. In this study, we have confirmed these down-regulated genes (including cytokines, chemokines, and p53 gene family members) as bona fide downstream transcriptional targets of TAZ. By using human breast and lung epithelial cells, we have further characterized ΔNp63, a p53 gene family member, and shown that TAZ suppresses ΔNp63 mRNA, protein expression, and promoter activity through interaction with the transcription factor TEAD. We also show that TEAD can inhibit ΔNp63 promoter activity and that TAZ can directly interact with ΔNp63 promoter-containing TEAD binding sites. Finally, we provide functional evidence that down-regulation of ΔNp63 by TAZ may play a role in regulating cell migration. Altogether, this study provides novel evidence that the Hippo component TAZ can function as a co-repressor and regulate biological functions by negatively regulating downstream cellular genes.

Since the metastatic progression of cancers is often fatal with limited treatment options, understanding the mechanism of metastasis is imperative for designing novel and targeted therapies. TAZ has been identified as a novel oncogene in both breast and lung cancers and is inhibited by the Hippo signaling pathway. In this study we provide convincing evidence that overexpression of TAZ in a mammary epithelial cell line, MCF10A, leads to enhanced cell migration - a fundamental characteristic of the metastatic progression of cancers. In addition, we identified the secreted growth factor BMP4 as a mediator of TAZ-induced cell migration. TAZ induces BMP4 transcription through the TEAD family of transcription factors, which mediate BMP4 promoter activation through binding to TEAD response element 1 (TRE1). Importantly, BMP4 activation by TAZ also enhances signaling downstream of TAZ, in particular, promoting Smad1/5 intracellular signaling. Functionally, short hairpin RNA-mediated knockdown of BMP4 rescued TAZ-induced cell migration. Our findings have identified a novel TAZ/TEAD/BMP4 signaling axis responsible for cell migration, with future implications in the development of targeted therapeutics for metastatic breast cancers.

Taxol (paclitaxel) resistance represents a major challenge in breast cancer treatment. The TAZ (transcriptional co-activator with PDZ-binding motif) oncogene is a major component of the novel Hippo–LATS signaling pathway and a transcriptional coactivator that interacts with and activates multiple transcription factors to regulate various biological processes. Here, we report that elevated levels of TAZ found in human breast cancer cells are responsible for their resistance to Taxol. DNA microarray analysis identified the oncogenes Cyr61 and CTGF as downstream transcriptional targets of TAZ. Short hairpin RNA–mediated knockdown of both Cyr61 and CTGF reversed TAZ-induced Taxol resistance in breast cancer cells. Interaction of TAZ with the TEAD family of transcription factors was essential for TAZ to activate the Cyr61/CTGF promoters and to induce Taxol resistance. Our findings define the TAZ-TEAD-Cyr61/CTGF signaling pathway as an important modifier of the Taxol response in breast cancer cells, as well as highlighting it as a novel therapeutic target to treat drug-resistant breast cancers that arise commonly at advanced stages of disease. Cancer Res; 71(7); 2728–38. ©2011 AACR.

Cutaneous melanoma is an aggressive form of human skin cancer characterized by high metastatic potential and poor prognosis. To better understand the role of microRNAs (miRNAs) in melanoma, the expression of 470 miRNAs was profiled in tissue samples from benign nevi and metastatic melanomas. We identified 31 miRNAs that were differentially expressed (13 up-regulated and 18 down-regulated) in metastatic melanomas relative to benign nevi. Notably, miR-193b was significantly down-regulated in the melanoma tissues examined. To understand the role of miR-193b in melanoma, functional studies were undertaken. Overexpression of miR-193b in melanoma cell lines repressed cell proliferation. Gene expression profiling identified 314 genes down-regulated by overexpression of miR-193b in Malme-3M cells. Eighteen of these down-regulated genes, including cyclin D1 (CCND1), were also identified as putative miR-193b targets by TargetScan. Overexpression of miR-193b in Malme-3M cells down-regulated CCND1 mRNA and protein byor = 50%. A luciferase reporter assay confirmed that miR-193b directly regulates CCND1 by binding to the 3'untranslated region of CCND1 mRNA. These studies indicate that miR-193b represses cell proliferation and regulates CCND1 expression and suggest that dysregulation of miR-193b may play an important role in melanoma development.

Since cancer is one of the leading causes of death worldwide, there is an urgent need to find better treatments. Currently, the use of chemotherapeutics remains the predominant option for cancer therapy. However, one of the major obstacles for successful cancer therapy using these chemotherapeutics is that patients often do not respond or eventually develop resistance after initial treatment. Therefore identification of genes involved in chemotherapeutic response is critical for predicting tumour response and treating drug-resistant cancer patients. A group of genes commonly lost or inactivated are tumour suppressor genes, which can promote the initiation and progression of cancer through regulation of various biological processes such as cell proliferation, cell death and cell migration/invasion. Recently, mounting evidence suggests that these tumour suppressor genes also play a very important role in the response of cancers to a variety of chemotherapeutic drugs. In the present review, we will provide a comprehensive overview on how major tumour suppressor genes [Rb (retinoblastoma), p53 family, cyclin-dependent kinase inhibitors, BRCA1 (breast-cancer susceptibility gene 1), PTEN (phosphatase and tensin homologue deleted on chromosome 10), Hippo pathway, etc.] are involved in chemotherapeutic drug response and discuss their applications in predicting the clinical outcome of chemotherapy for cancer patients. We also propose that tumour suppressor genes are critical chemotherapeutic targets for the successful treatment of drug-resistant cancer patients in future applications.

MiRNAs regulate pathways associated with differentiation, proliferation, apoptosis, among others, miRNAs missregulation may contribute to malignant transformation. Although melanoma is one of the rarest dermatological cancers, it is responsible for the greatest number of skin cancer-related deaths. In that context, our aim is to identify miRNAs that could be involved with melanoma progression. Using a cellular murine model of melanoma development, we identified the miR-138 as an interesting target of studying. In this model, four cell lines (melan-a, 4C, 4C11- and 4C11+) mimics the distinct steps of human melanoma genesis. miR-138 is codified from two different regions on the genome and they were named according to their origin. miR-138/1 is coded from chromosome 9 in mouse (corresponding to 3, in humans) while miR138/2 is coded from chromosome 8 in mouse (equivalent to 16, in humans). Despite the mature sequence of these two miRNAs being exactly the same, the flanking regions that form the precursors are distinct. miR-138 coded from chromosome 8 but not from chromosome 9 promotes cells to acquire a more aggressive phenotype. When we transfected the tumorigenic but non-metastatic cell line 4C11- with the genomic DNA corresponding to miR-138/2, it promotes the increase of proliferation, migration, colony formation and resistance to anoikis by this cell line compared to its untransfected counterpart 4C11-. In vivo assays showed that 4C11- miR-138/2 is able to form fast-growing tumors and pulmonary metastasis. None of these phenotypic changes was observed in 4C11- cells overexpressing the miR-138 coded from chromosome 9. We also validated p53 (rarely mutated in melanomas) as a target of miR-138 coded from chromosome 8, but not from chromosome 9. Therefore, we hypothesized that the sequence of precursor miRNA (pre-miR) could be involved with the direction of mature miRNA to the mRNA target. Moreover, EZH2, validated as a target of miR-138, is downregulated only in 4C11- cells overexpressing the miR-138/1, emphasizing even more the importance of the precursor sequence in the regulation of the mRNA target. miR-138 expression in melanocytic lesion were evaluated as well. We observed increased expression of miR-138 in primary and metastatic human melanoma samples related to benign nevi. Therefore, in this work we showed that miR-138 may contribute to melanoma genesis and is capable to regulate the expression of p53. Moreover, we suggest for the first time that the precursor sequence of miRNA can direct the mature miRNA to mRNA targets. Supported by CNPq and FAPESP Citation Format: Adriana Taveira Da Cruz, Aline Hunger, Genevieve Paré, Dulcie Lai, Fabiana Henriques Machado Melo, Bryan Eric Strauss, Victor Tron, Miriam Galvonas Jasiulionis. The precursor miR-138/2, but not miR-138/1, targets p53 mRNA and contributes to the acquisition of melanoma metastatic phenotype: Are the miRNAs precursors important to direct mature miRNA to mRNA targets. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1458. doi:10.1158/1538-7445.AM2014-1458

Concern over the tremendous loss of genetic diversity among many of our most important crops has prompted major efforts to preserve seed stocks derived from cultivated species and their wild relatives. Arabidopsis thaliana propagates mainly by self-fertilizing, and therefore, like many crop plants, theoretically has a limited potential for producing genetically diverse offspring. Despite this, inbreeding has persisted in Arabidopsis for over a million years suggesting that some underlying adaptive mechanism buffers the deleterious consequences of this reproductive strategy. Using presence-absence molecular markers we demonstrate that single Arabidopsis plants can have multiple genotypes. Sequence analyses reveal single nucleotide changes, loss of sequences and, surprisingly, acquisition of unique genomic insertions. Estimates based on quantitative analyses suggest that these genetically discordant sectors are very small but can have a complex genetic makeup. In ruling out more trivial explanations for these data, our findings raise the possibility that intrinsic drivers of genetic variation are responsible for the targeted sequence changes we detect. Given the evolutionary advantage afforded to populations with greater genetic diversity, we hypothesize that organisms that primarily self-fertilize or propagate clonally counteract the genetic cost of such reproductive strategies by leveraging a cryptic reserve of extra-genomic information.

TAZ functions as a transcriptional co-activator critical for tissue development, mesenchymal stem cell differentiation, and embryonic stem cell renewal. Recently, TAZ has been identified as a major downstream component of the novel Hippo-LATS tumor suppressor pathway, where TAZ over-expression in immortalized mammary epithelial cells, MCF10A, leads to enhanced cell proliferation and migration, transformation, EMT, and resistance to the chemotherapeutic paclitaxel. However, the molecular mechanisms underlying these TAZ-induced phenotypes are largely unknown. In this study, we aimed to identify downstream targets responsible for TAZ-induced phenotypes. Through screening a 44K human genome microarray we have identified many potential targets, one of which is bone morphogenetic protein 4 (BMP4), a cytokine that regulates important processes such as cell proliferation, migration, and EMT. By qRT-PCR, western blotting, and an ELISA assay we verified that BMP4 is upregulated at the mRNA, protein, and secreted levels, respectively, in TAZ overexpressing MCF10A cells (MCF10A-TAZ) compared to control cells (MCF10A-WPI). To investigate whether BMP4 is a direct transcriptional target of TAZ, we used a dual luciferase assay to measure BMP4 promoter activity. When co-transfected with TAZ and TEAD4, a transcription factor mediating TAZ transactivation, BMP4 promoter activity was significantly increased. This TAZ/TEAD interaction is critical for BMP4 transcriptional activation as interaction mutants of TAZ and TEAD4 (TAZα72 and TEAD4-Y429H) both fail to activate BMP4 promotor activity. Significantly, we have identified two TEAD Response Elements (TRE) located in the BMP4 promoter where mutation of TRE1 alone completely abolished activation of BMP4 promoter activity by TAZ/TEAD4 and mutation of TRE2 had no effect. This suggests that TRE1 is responsible for TAZ/TEAD activation of BMP4. Through microarray analysis, we have identified many secreted cytokines leading us to suspect that they may promote EMT. Strikingly, MCF10A cells continuously treated with conditioned media from MCF10A-TAZ or exogenous BMP4 induced an obvious EMT phenotype, suggesting that BMP4 may be responsible for TAZ-induced EMT. Moreover, phosphorylation of Smad1,5 (pSmad1,5), two major mediators of BMP4 function, was also significantly increased in MCF10A-TAZ, but was completely abolished when MCF10A-TAZ cells were treated with Noggin, a BMP4 specific inhibitor, demonstrating that activation of Smad is specifically due to BMP4. Our results provide convincing evidence that BMP4 is a novel transcriptional target of TAZ, which may mediate the TAZ-induced EMT phenotype and thereby identify a novel TAZ/TEAD/BMP4/Smad1,5 signaling axis. Further functional characterization of this pathway will greatly facilitate our understanding of the roles of TAZ in the metastatic progression of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3247. doi:1538-7445.AM2012-3247