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African American (AA) women are twice as likely to develop triple-negative breast cancer (TNBC) as women of European descent. Additionally, AA women with TNBC present a much more aggressive disease course than their European American (EA) counterparts. Thus, there is an unmet clinical need to identify race-specific biomarkers and improve survival outcomes in AA patients with TNBC. The minus-end directed microtubule motor protein kinesin family member C1 (KIFC1) promotes centrosome clustering and chromosomal instability and is often overexpressed in TNBC. Previous findings suggest that KIFC1 plays a role in cell proliferation and migration in TNBC cells from AAs and that the levels of nuclear KIFC1 (nKIFC1) are particularly high in AA patients with TNBC. The nuclear localization of KIFC1 in interphase may underlie its previously unrecognized race-specific association. In this study, we found that in TNBC cells derived from AAs, nKIFC1 interacted with the tumor suppressor myosin heavy chain 9 (MYH9) over EA cells. Treatment of AA TNBC cells with commercial inhibitors of KIFC1 and MYH9 disrupted the interaction between KIFC1 and MYH9. To characterize the racial differences in the KIFC1-MYH9-MYC axis in TNBC, we established homozygous KIFC1 knockout (KO) TNBC cell lines. KIFC1 KO significantly inhibited proliferation, migration, and invasion in AA TNBC cells but not in EA TNBC cells. RNA sequencing analysis showed significant downregulation of genes involved in cell migration, invasion, and metastasis upon KIFC1 KO in TNBC cell lines from AAs compared to those from EAs. These data indicate that mechanistically, the role of nKIFC1 in driving TNBC progression and metastasis is stronger in AA patients than in EA patients, and that KIFC1 may be a critical therapeutic target for AA patients with TNBC.
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Cinesinas , Cadeias Pesadas de Miosina , Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/etnologia , Neoplasias de Mama Triplo Negativas/metabolismo , Cinesinas/genética , Cinesinas/metabolismo , Feminino , Linhagem Celular Tumoral , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Proliferação de Células/genética , Movimento Celular/genética , Negro ou Afro-Americano/genética , População Branca/genética , Ligação ProteicaRESUMO
Rationale: Within chronic obstructive pulmonary disease (COPD), emphysema is characterized by a significant yet partially understood B cell immune component. Objectives: To characterize the transcriptomic signatures from lymphoid follicles (LFs) in ever-smokers without COPD and patients with COPD with varying degrees of emphysema. Methods: Lung sections from 40 patients with COPD and ever-smokers were used for LF proteomic and transcriptomic spatial profiling. Formalin- and O.C.T.-fixed lung samples obtained from biopsies or lung explants were assessed for LF presence. Emphysema measurements were obtained from clinical chest computed tomographic scans. High-confidence transcriptional target intersection analyses were conducted to resolve emphysema-induced transcriptional networks. Measurements and Main Results: Overall, 115 LFs from ever-smokers and Global Initiative for Chronic Obstructive Lung Disease (GOLD) 1-2 and GOLD 3-4 patients were analyzed. No LFs were found in never-smokers. Differential gene expression analysis revealed significantly increased expression of LF assembly and B cell marker genes in subjects with severe emphysema. High-confidence transcriptional analysis revealed activation of an abnormal B cell activity signature in LFs (q-value = 2.56E-111). LFs from patients with GOLD 1-2 COPD with emphysema showed significantly increased expression of genes associated with antigen presentation, inflammation, and B cell activation and proliferation. LFs from patients with GOLD 1-2 COPD without emphysema showed an antiinflammatory profile. The extent of centrilobular emphysema was significantly associated with genes involved in B cell maturation and antibody production. Protein-RNA network analysis showed that LFs in emphysema have a unique signature skewed toward chronic B cell activation. Conclusions: An off-targeted B cell activation within LFs is associated with autoimmune-mediated emphysema pathogenesis.
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Enfisema , Linfadenopatia , Doença Pulmonar Obstrutiva Crônica , Enfisema Pulmonar , Humanos , Enfisema Pulmonar/diagnóstico por imagem , Enfisema Pulmonar/genética , Proteômica , Perfilação da Expressão GênicaRESUMO
Precision and personalized therapeutics have witnessed significant advancements in technology, revolutionizing the capabilities of laboratories to generate vast amounts of genetic data. Coupled with computational resources for analysis and interpretation, and integrated with various other types of data, including genomic data, electronic medical health (EMH) data, and clinical knowledge, these advancements support optimized health decisions. Among these technologies, next-generation sequencing (NGS) stands out as a transformative tool in the field of cancer treatment, playing a crucial role in precision oncology. NGS-based workflows are employed across a range of applications, including gene panels, exome sequencing, and whole-genome sequencing, supporting comprehensive analysis of the entire cancer genome, including mutations, copy number variations, gene expression profiles, and epigenetic modifications. By utilizing the power of NGS, these workflows contribute to enhancing our understanding of disease mechanisms, diagnosis confirmation, identifying therapeutic targets, and guiding personalized treatment decisions. This manuscript explores the diverse applications of NGS in cancer treatment, highlighting its significance in guiding diagnosis and treatment decisions, identifying therapeutic targets, monitoring disease progression, and improving patient outcomes.
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Neoplasias , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/terapia , Análise de Sequência de DNA , Variações do Número de Cópias de DNA , Patologia Molecular , Medicina de Precisão , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
RNA splicing is the fundamental process that brings diversity at the transcriptome and proteome levels. The spliceosome complex regulates minor and major processes of RNA splicing. Aberrant regulation is often associated with different diseases, including diabetes, stroke, hypertension, and cancer. In the majority of cancers, dysregulated alternative RNA splicing (ARS) events directly affect tumor progression, invasiveness, and often lead to poor survival of the patients. Alike the rest of the gastrointestinal malignancies, in hepatocellular carcinoma (HCC), which alone contributes to ~ 75% of the liver cancers, a large number of ARS events have been observed, including intron retention, exon skipping, presence of alternative 3'-splice site (3'SS), and alternative 5'-splice site (5'SS). These events are reported in spliceosome and non-spliceosome complexes genes. Molecules such as MCL1, Bcl-X, and BCL2 in different isoforms can behave as anti-apoptotic or pro-apoptotic, making the spliceosome complex a dual-edged sword. The anti-apoptotic isoforms of such molecules bring in resistance to chemotherapy or cornerstone drugs. However, in contrast, multiple malignant tumors, including HCC that target the pro-apoptotic favoring isoforms/variants favor apoptotic induction and make chemotherapy effective. Herein, we discuss different splicing events, aberrations, and antisense oligonucleotides (ASOs) in modulating RNA splicing in HCC tumorigenesis with a possible therapeutic outcome.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Processamento Alternativo , Carcinoma Hepatocelular/genética , Humanos , Íntrons , Neoplasias Hepáticas/genética , Isoformas de Proteínas/genética , Sítios de Splice de RNARESUMO
Thyroid receptor-interacting protein 13 (TRIP13), a protein of the AAA-ATPase family, is upregulated in various human cancers, including colorectal cancer (CRC). This study focused on the inhibition of TRIP13-induced CRC progression and signalling by DCZ0415, a small molecule targeting TRIP13. It demonstrated potent antitumour activity in TRIP13-deregulated cancer cell lines, regardless of their p53, KRAS, BRAF, epidermal growth factor receptor or microsatellite instability status. The treatment of CRC cells with DCZ0415 resulted in decreased cell proliferation, induced cell cycle arrest in the G2-M phase and increased apoptosis. DCZ0415 diminished xenograft tumour growth and metastasis of CRC in immunocompromised mice. DCZ0415 reduced expression of fibroblast growth factor receptor 4 (FGFR4), signal transducer and activator of transcription 3 (STAT3), and proteins associated with the epithelial-mesenchymal transition and nuclear factor kappa B (NF-κB) pathways in cells and xenografts exhibiting high expression of TRIP13. Additionally, DCZ0415 decreased cyclin D1, ß-catenin and T-cell factor 1, leading to the inactivation of the Wnt/ß-catenin pathway. In a syngeneic CRC model, DCZ0415 treatment induced an immune response by decreasing PD1 and CTLA4 levels and increasing granzyme B, perforin and interferon gamma. In sum, DCZ04145 inhibits the TRIP13-FGFR4-STAT3 axis, inactivates NF-κB and Wnt/ß-catenin signalling, activates antitumour immune response and reduces the progression and metastasis of CRC. This study provides a rationale to evaluate DCZ0415 clinically for the treatment of a subset of CRCs that exhibit dysregulated TRIP13 and FGFR4.
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Neoplasias Colorretais , beta Catenina , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/genética , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , NF-kappa B/metabolismo , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Fator de Transcrição STAT3/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismoRESUMO
Cancer genomic, transcriptomic, and proteomic profiling has generated extensive data that necessitate the development of tools for its analysis and dissemination. We developed UALCAN to provide a portal for easy exploring, analyzing, and visualizing these data, allowing users to integrate the data to better understand the gene, proteins, and pathways perturbed in cancer and make discoveries. UALCAN web portal enables analyzing and delivering cancer transcriptome, proteomics, and patient survival data to the cancer research community. With data obtained from The Cancer Genome Atlas (TCGA) project, UALCAN has enabled users to evaluate protein-coding gene expression and its impact on patient survival across 33 types of cancers. The web portal has been used extensively since its release and received immense popularity, underlined by its usage from cancer researchers in more than 100 countries. The present manuscript highlights the task we have undertaken and updates that we have made to UALCAN since its release in 2017. Extensive user feedback motivated us to expand the resource by including data on a) microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and promoter DNA methylation from TCGA and b) mass spectrometry-based proteomics from the Clinical Proteomic Tumor Analysis Consortium (CPTAC). UALCAN provides easy access to pre-computed, tumor subgroup-based gene/protein expression, promoter DNA methylation status, and Kaplan-Meier survival analyses. It also provides new visualization features to comprehend and integrate observations and aids in generating hypotheses for testing. UALCAN is accessible at http://ualcan.path.uab.edu.
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Neoplasias , Proteômica , Metilação de DNA , Análise de Dados , Perfilação da Expressão Gênica , Genômica , Humanos , Neoplasias/metabolismoRESUMO
Lung cancer is the leading cause of cancer-related deaths globally and is histologically defined as either small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC), with the latter accounting for 80% of all lung cancers. The 5-year overall survival rate for lung cancer patients is low as it is often discovered at advanced stages when potential cure by surgical resection is no longer an option. To identify a biomarker and target for lung cancer, we performed analysis of multiple datasets of lung cancer gene expression data. Our analyses indicated that the collagen-modifying enzyme Prolyl 4-Hydroxylase Subunit Alpha 1 (P4HA1) is overexpressed in NSCLC. Furthermore, our investigation found that overexpression of enzymes involved in this pathway predicts poor outcome for patients with lung adenocarcinoma. Our functional studies using knockdown strategies in lung cancer cell lines in vitro indicated that P4HA1 is critical for lung cancer growth, migration, and invasion. Additionally, diethyl pythiDC (PythiDC), a small molecule inhibitor, decreased the malignant phenotypes of lung cancer cells. Moreover, we found that miR-124 regulates and targets P4HA1 in lung cancer cells. Thus, our study suggests that collagen-modifying enzymes play an important role in lung cancer aggressiveness. Furthermore, our studies showed that P4HA1 is required for lung cancer cell growth and invasion, suggesting its potential as a valid therapeutic target in lung adenocarcinoma.
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PURPOSE: Triple-negative breast cancer (TNBC) patients of various ethnic groups often have discrete clinical presentations and outcomes. Women of African descent have a disproportionately higher chance of developing TNBCs. The aim of the current study was to establish the transcriptome of TNBCs from Kenyan (KE) women of Bantu origin and compare it to those TNBCs of African-Americans (AA) and Caucasians (CA) for identifying KE TNBC-specific molecular determinants of cancer progression and potential biomarkers of clinical outcomes. PATIENTS AND METHODS: Pathology-confirmed TNBC tissues from Kenyan women of Bantu origin (n = 15) and age and stage range matched AA (n = 19) and CA (n = 23) TNBCs of patients from Alabama were included in this study. RNA was isolated from paraffin-embedded tissues, and expression was analyzed by RNA sequencing. RESULTS: At clinical presentation, young KE TNBC patients have tumors of higher stages. Differential expression analysis identified 160 up-regulated and 178 down-regulated genes in KE TNBCs compared to AA and CA TNBCs. Validation analyses of the TCGA breast cancer data identified 45 KE TNBC-specific genes that are involved in the apoptosis (ACTC1, ERCC6 and CD14), cell proliferation (UHRF2, KDM4C, UHMK1, KCNH5, KRT18, CSF1R and S100A13), and Wnt signaling (BCL9L) pathways. CONCLUSIONS: In this study, we identified biomarkers that are specific for KE TNBC patients of Bantu origin. Further study with a larger sample size of matched tumors could confirm our findings. If biologically confirmed, these molecular determinants could have clinical and biological implications and serve as targets for development of personalized therapeutics for KE TNBC patients.
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BACKGROUND: Metaplastic carcinoma is an aggressive, triple-negative breast cancer (TNBC) with differentiation towards squamous, spindle, or mesenchymal cell types. The molecular underpinnings of the histological subtypes are unclear. Our lab discovered a cytoplasmic function of EZH2, a transcriptional repressor, whereby pEZH2 T367 binds to cytoplasmic proteins in TNBC cells and enhances invasion and metastasis. Here, we investigated the expression and subcellular localization of pEZH2 T367 protein in metaplastic carcinomas. METHODS: Thirty-five metaplastic carcinomas (17 squamous, 10 mesenchymal, and 8 spindle) were evaluated and immunostained with anti-pEZH2 T367. We analyzed staining intensity (score 1-4), subcellular localization (nuclear/cytoplasmic), and localization within the tumor (center/invasive edge). Protein expression of pEZH2 T367-binding partners was measured from a quantitative multiplex proteomics analysis performed in our lab. RESULTS: Cytoplasmic pEZH2 T367 was significantly upregulated in squamous (14 of 17, 82%) compared to mesenchymal (4 of 10, 40%) and spindle (2 of 6, 33%) subtypes (p = 0.011). Twenty-five of 34 (73%) tumors with available tumor-normal interface showed accentuated cytoplasmic pEZH2 T367 at the infiltrative edge. Cytoplasmic pEZH2 T367 was upregulated in 9 of 10 (90%) tumors with lymph node metastasis (p = 0.05). Bioinformatics analyses identified an EZH2 protein network in metaplastic carcinomas (p value: < 1.0e-16). Using quantitative proteomics, we found significantly increased expression of cytoplasmic EZH2-binding partners in squamous compared to spindle and mesenchymal subtypes. CONCLUSIONS: pEZH2 T367 expression and subcellular localization may be useful to distinguish metaplastic carcinoma subtypes. pEZH2 T367 may play a role in the histological diversity and behavior of these tumors.
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Carcinoma de Células Escamosas/metabolismo , Citoplasma/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteoma , Neoplasias de Mama Triplo Negativas/metabolismo , Estudos de Coortes , Biologia Computacional/métodos , Feminino , Humanos , Imuno-Histoquímica/métodos , Metástase Linfática , Pessoa de Meia-Idade , Fosforilação , Prognóstico , Proteômica/métodos , Neoplasias de Mama Triplo Negativas/classificação , Regulação para CimaRESUMO
Exposure of rats to 2-chloroethyl ethyl sulfide (CEES), an analog of sulfur mustard, can cause acute lung injury (ALI), resulting in increased inflammation and coagulation and altered levels of plasma microRNAs (miRNAs). Rats were exposed to aerosolized CEES and euthanized 12 h later for collection of tissue and plasma. Profiling of miRNAs in plasma, using a TaqMan-based RT-PCR array, revealed 14 differentially expressed miRNAs. Target gene prediction and pathway analysis revealed miRNA-mediated regulation of organismal injury, inflammation, and respiratory diseases. miR-140-5p, a marker of ALI, was downregulated in the plasma, lung, liver, and kidney of CEES-exposed rats, with a concomitant increase in the expression of the inflammation markers IL-6 and IL-1α and the coagulation marker tissue factor (F3). Exposure of rat airway epithelial cells (RL-65) to CEES (0.5 mM) caused cell death and a decrease in miR-140-5p both in cells and media supernatant. This was accompanied by an increase in cellular mRNA levels of IL-6, IL-1α, and F3, as well as FGF9 and EGR2, putative targets of miR-140. Knockdown of miR-140 by specific oligos in RL-65 cells mimicked the in vivo CEES-mediated effects, leading to significantly increased mRNA levels of IL-6, IL-1α, F3, FGF9, and EGR2. Our study identifies miR-140-5p as a mediator of CEES-induced ALI, which could potentially be targeted for therapy.
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Lesão Pulmonar Aguda , Coagulação Sanguínea/efeitos dos fármacos , Substâncias para a Guerra Química/toxicidade , MicroRNAs/metabolismo , Gás de Mostarda/análogos & derivados , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/metabolismo , Animais , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Fator 9 de Crescimento de Fibroblastos/genética , Fator 9 de Crescimento de Fibroblastos/metabolismo , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/metabolismo , Interleucina-1alfa/genética , Interleucina-1alfa/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , MicroRNAs/genética , Gás de Mostarda/toxicidade , Ratos , Ratos Sprague-DawleyRESUMO
Hepatocellular carcinoma (HCC), the most malignant form of primary liver cancer, is the fourth most prevalent cause of cancer mortality globally. It was recently discovered that the dietary fermentable fiber, inulin, can reprogram the murine liver to favor HCC development in a gut microbiota-dependent manner. Determining the molecular pathways that are either over expressed or repressed during inulin-induced HCC would provide a platform of potential therapeutic targets. In the present study, we have combined analysis of the novel inulin-induced HCC murine model and human HCC samples to identify differentially expressed genes (DEGs) in hepatocarcinogenesis. Hepatic transcriptome profiling revealed that there were 674 DEGs in HCC mice compared to mice safeguarded from HCC. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis uncovered enrichment in ECM-receptor interaction, steroid hormone biosynthesis, PPAR signaling pathway, focal adhesion and protein digestion and absorption during inulin-induced HCC. Tandem mass tag based quantitative, multiplexed proteomic analysis delineated 57 differentially expressed proteins, where the over-expressed proteins were associated with cell adhesion molecules, valine, leucine and isoleucine degradation and ECM-receptor interaction. After obtaining the human orthologs of the mouse genes, we did a comparison analysis to level 3 RNA-seq data found in the Cancer Genome Atlas (TCGA) database, corresponding to human HCC (n = 361) and healthy liver (n = 50) samples. Out of the 549 up-regulated and 68 down-regulated human orthologs identified, 142 genes (137 significantly over-expressed and 5 significantly under-expressed) were associated with human HCC. Using univariate survival analysis, we found 27 over-expressed genes involved in cell-cell adhesion and cell division that were associated with poor HCC patient survival. Overall, the genetic and proteomics signatures highlight potential underlying mechanisms in inulin-induced HCC and support that this murine HCC model is human relevant.
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Carcinoma Hepatocelular/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/patologia , Animais , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidade , Moléculas de Adesão Celular/metabolismo , Modelos Animais de Doenças , Ontologia Genética , Humanos , Insulina/toxicidade , Estimativa de Kaplan-Meier , Fígado/metabolismo , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Proteômica , Transdução de Sinais , Receptor 5 Toll-Like/deficiência , Receptor 5 Toll-Like/genética , TranscriptomaRESUMO
Functional fermentable fibers are considered essential for a healthy diet. Recently, we demonstrated that gut microbiota dysbiotic mice fed an inulin-containing diet (ICD) developed hepatocellular carcinoma (HCC) within 6 mo. In particular, a subset of Toll-like receptor 5-deficient (T5KO) mice prone to HCC exhibited rapid onset of hyperbilirubinemia (HB) and cholemia; these symptoms provide rationale that ICD induces cholestasis. Our objective in the present study was to determine whether inulin-fed T5KO-HB mice exhibit other known consequences of cholestasis, including essential fatty acid and fat-soluble vitamin deficiencies. Here, we measured hepatic fatty acids and serum vitamin A and D levels from wild-type (WT), T5KO low bilirubin (LB) and T5KO-HB mice fed ICD for 4 wk. Additionally, hepatic RNAseq and proteomics were performed to ascertain other metabolic alterations. Compared with WT and T5KO-LB, T5KO-HB mice exhibited steatorrhea, i.e., ~50% increase in fecal lipids. This could contribute to the significant reduction of linoleate in hepatic neutral lipids in T5KO-HB mice. Additionally, serum vitamins A and D were ~50% reduced in T5KO-HB mice, which was associated with metabolic compromises. Overall, our study highlights that fermentable fiber-induced cholestasis is further characterized by depletion of macro-and micronutrients.NEW & NOTEWORTHY Feeding a dietary, fermentable fiber diet to a subset of Toll-like receptor 5 deficient (T5KO) mice induces early onset hyperbilirubinemia and cholemia that later manifests to hepatocellular carcinoma (HCC). Our study highlights that fermentable fiber-induced cholestasis is characterized with modest macro- and micronutrient deficiencies that may further contribute to hepatic biliary disease. Compared with chemical induction, immunization, surgery, or genetic manipulation, these findings provide a novel approach to study the cholestatic subtype of HCC.
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Fibras na Dieta , Fígado Gorduroso/metabolismo , Absorção Intestinal , Inulina , Metabolismo dos Lipídeos , Fígado/metabolismo , Síndromes de Malabsorção/metabolismo , Receptor 5 Toll-Like/deficiência , Deficiência de Vitamina A/metabolismo , Deficiência de Vitamina D/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Colestase/genética , Colestase/metabolismo , Colestase/patologia , Modelos Animais de Doenças , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Fermentação , Fígado/patologia , Síndromes de Malabsorção/genética , Síndromes de Malabsorção/patologia , Masculino , Camundongos Knockout , Receptor 5 Toll-Like/genética , Deficiência de Vitamina A/genética , Deficiência de Vitamina D/genéticaRESUMO
Cancer cells utilize vitamin folate to fulfill their excessive demand for nucleotides and amino acids. Dihydrofolate reductase (DHFR), an enzyme involved in folate metabolism converts dihydrofolate into tetrahydrofolate, which is required for the de novo synthesis of purines, and certain amino acids. DHFR inhibitors are used as a chemotherapeutic agent. Cancer sequencing analysis has identified additional enzymes in folate metabolism that are dysregulated in cancer. Methylenetetrahydrofolate dehydrogenase 1 like (MTHFD1L), one such enzyme is overexpressed in bladder cancer. MTHFD1L is a mitochondrial enzyme involved in the folate cycle by catalyzing the reaction of formyl-tetrahydrofolate to formate and tetrahydrofolate (THF). THF is crucial for de novo purine and thymidylate synthesis and is also involved in the regeneration of methionine. Cancer cells rely on purines derived from the de novo pathway for the nucleotides whereas normal cells favor the salvage pathway. In this study we examined MTHFD1L expression in bladder cancer. By using publicly available cancer transcriptome data analysis web-portal UALCAN, we found overexpression of MTHFD1L in bladder cancer and expression was associated with overall survival. RT-PCR and immunoblot analysis confirmed the overexpression of MTHFD1L in muscle invasive bladder cancer tissues compared to normal urothelium. Furthermore, our investigations suggested a critical role for MTHFD1L in bladder cancer cell proliferation, colony formation and invasion. Thus, in this study, we show the significance of the folate metabolic enzyme MTHFD1L in aggressive bladder cancers and suggest that being an enzyme, MTHFD1L serves as a valuable therapeutic target.
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BACKGROUND: Recent microarray and sequencing studies of prostate cancer showed multiple molecular alterations during cancer progression. It is critical to evaluate these molecular changes to identify new biomarkers and targets. We performed analysis of glycine-N-acyltransferase like 1 (GLYATL1) expression in various stages of prostate cancer in this study and evaluated the regulation of GLYATL1 by androgen. METHOD: We performed in silico analysis of cancer gene expression profiling and transcriptome sequencing to evaluate GLYATL1 expression in prostate cancer. Furthermore, we performed immunohistochemistry using specific GLYATL1 antibody using high-density prostate cancer tissue microarray containing primary and metastatic prostate cancer. We also tested the regulation of GLYATL1 expression by androgen and ETS transcription factor ETV1. In addition, we performed RNA-sequencing of GLYATL1 modulated prostate cancer cells to evaluate the gene expression and changes in molecular pathways. RESULTS: Our in silico analysis of cancer gene expression profiling and transcriptome sequencing we revealed an overexpression of GLYATL1 in primary prostate cancer. Confirming these findings by immunohistochemistry, we show that GLYATL1 is overexpressed in primary prostate cancer compared with metastatic prostate cancer and benign prostatic tissue. Low-grade cancers had higher GLYATL1 expression compared to high-grade prostate tumors. Our studies showed that GLYATL1 is upregulated upon androgen treatment in LNCaP prostate cancer cells which harbors ETV1 gene rearrangement. Furthermore, ETV1 knockdown in LNCaP cells showed downregulation of GLYATL1 suggesting potential regulation of GLYATL1 by ETS transcription factor ETV1. Transcriptome sequencing using the GLYATL1 knockdown prostate cancer cell lines LNCaP showed regulation of multiple metabolic pathways. CONCLUSIONS: In summary, our study characterizes the expression of GLYATL1 in prostate cancer and explores the regulation of its regulation in prostate cancer showing role for androgen and ETS transcription factor ETV1. Future studies are needed to decipher the biological significance of these findings.
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Aciltransferases/metabolismo , Neoplasias da Próstata/enzimologia , Aciltransferases/genética , Androgênios/farmacologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Masculino , Próstata/enzimologia , Neoplasias da Próstata/patologia , Análise Serial de Tecidos , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Sequenciamento do ExomaRESUMO
We present the functional characterization of a pseudogene associated recurrent gene fusion in prostate cancer. The fusion gene KLK4-KLKP1 is formed by the fusion of the protein coding gene KLK4 with the noncoding pseudogene KLKP1. Screening of a cohort of 659 patients (380 Caucasian American; 250 African American, and 29 patients from other races) revealed that the KLK4-KLKP1 is expressed in about 32% of prostate cancer patients. Correlative analysis with other ETS gene fusions and SPINK1 revealed a concomitant expression pattern of KLK4-KLKP1 with ERG and a mutually exclusive expression pattern with SPINK1, ETV1, ETV4, and ETV5. Development of an antibody specific to KLK4-KLKP1 fusion protein confirmed the expression of the full-length KLK4-KLKP1 protein in prostate tissues. The in vitro and in vivo functional assays to study the oncogenic properties of KLK4-KLKP1 confirmed its role in cell proliferation, cell invasion, intravasation, and tumor formation. Presence of strong ERG and AR binding sites located at the fusion junction in KLK4-KLKP1 suggests that the fusion gene is regulated by ERG and AR. Correlative analysis of clinical data showed an association of KLK4-KLKP1 with lower preoperative PSA values and in young men (<50â¯years) with prostate cancer. Screening of patient urine samples showed that KLK4-KLKP1 can be detected noninvasively in urine. Taken together, we present KLK4-KLKP1 as a class of pseudogene associated fusion transcript in cancer with potential applications as a biomarker for routine screening of prostate cancer.
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Fusão Gênica , Proteínas de Fusão Oncogênica/genética , Neoplasias da Próstata/genética , Pseudogenes , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Embrião de Galinha , Regulação Neoplásica da Expressão Gênica , Loci Gênicos , Humanos , Calicreínas/química , Calicreínas/genética , Masculino , Gradação de Tumores , Proteínas de Fusão Oncogênica/química , Calicreínas Teciduais/química , Calicreínas Teciduais/genéticaRESUMO
BACKGROUND: Genome-wide repeat sequences, such as LINEs, SINEs and LTRs share a considerable part of the mammalian nuclear genomes. These repeat elements seem to be important for multiple functions including the regulation of transcription initiation, alternative splicing and DNA methylation. But it is not possible to study all repeats and, hence, it would help to short-list before exploring their potential functional significance via experimental studies and/or detailed in silico analyses. RESULT: We developed the 'Genomic Repeat Element Analyzer for Mammals' (GREAM) for analysis, screening and selection of potentially important mammalian genomic repeats. This web-server offers many novel utilities. For example, this is the only tool that can reveal a categorized list of specific types of transposons, retro-transposons and other genome-wide repetitive elements that are statistically over-/under-represented in regions around a set of genes, such as those expressed differentially in a disease condition. The output displays the position and frequency of identified elements within the specified regions. In addition, GREAM offers two other types of analyses of genomic repeat sequences: a) enrichment within chromosomal region(s) of interest, and b) comparative distribution across the neighborhood of orthologous genes. GREAM successfully short-listed a repeat element (MER20) known to contain functional motifs. In other case studies, we could use GREAM to short-list repetitive elements in the azoospermia factor a (AZFa) region of the human Y chromosome and those around the genes associated with rat liver injury. GREAM could also identify five over-represented repeats around some of the human and mouse transcription factor coding genes that had conserved expression patterns across the two species. CONCLUSION: GREAM has been developed to provide an impetus to research on the role of repetitive sequences in mammalian genomes by offering easy selection of more interesting repeats in various contexts/regions. GREAM is freely available at http://resource.ibab.ac.in/GREAM/.