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1.
Aging (Albany NY) ; 11(19): 8046-8047, 2019 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-31612868
2.
Clin Cancer Res ; 2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31662330

RESUMO

PURPOSE: Chromosomal instability (CIN) is a common phenomenon in colorectal cancer (CRC), but its role and underlying cause remain unknown. We have identified that mitotic regulator microtubule-associated protein 9 (MAP9) as a critical regulator of CIN in CRC. We thus studied the effect of MAP9 loss on CRC in Map9 knockout mice and in cell lines. EXPERIMENTAL DESIGN: We generated colon epithelial-specific Map9 knockout mice and evaluated CRC development. Effect of Map9 knockout on CRC progression was determined in chemical or ApcMin/+-induced CRC. Molecular mechanism of MAP9 was determined using spectral karyotyping (SKY), microtubule assays and whole genome sequencing (WGS). Clinical significance of MAP9 was examined in 141 CRC patients. RESULTS: Spontaneous colonic tumors (9.1%) were developed in colon epithelium-specific Map9 knockout mice at 17 months, but none was observed in wildtype littermates. Map9 deletion accelerated CRC formation both in ApcMin/+ mice and azoxymethane-treated mice, and reduced survival in ApcMin/+ mice. Mechanistically, MAP9 stabilized microtubules and mediated mitotic spindle assembly. MAP9 also maintained the spindle pole integrity and protected K-fibre from depolymerization at spindle poles. MAP9 loss induced severe mitosis failure, chromosome segregation errors and aneuploidy, leading to transformation of normal colon epithelial cells. WGS confirmed enhanced CIN in intestinal tumors from Map9 knockout ApcMin/+ mice. In CRC patients, MAP9 was frequently silenced and its down-regulation was associated with poor survival. CONCLUSIONS: MAP9 is a microtubule stabilizer that contributes to spindle stability and inhibits colorectal tumorigenesis, supporting the role of MAP9 as a tumor suppressor for preventing CIN in CRC.

3.
Genome Res ; 29(11): 1878-1888, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31548359

RESUMO

Mitochondria are involved in a number of diverse cellular functions, including energy production, metabolic regulation, apoptosis, calcium homeostasis, cell proliferation, and motility, as well as free radical generation. Mitochondrial DNA (mtDNA) is present at hundreds to thousands of copies per cell in a tissue-specific manner. mtDNA copy number also varies during aging and disease progression and therefore might be considered as a biomarker that mirrors alterations within the human body. Here, we present a new quantitative, highly sensitive droplet digital PCR (ddPCR) method, droplet digital mitochondrial DNA measurement (ddMDM), to measure mtDNA copy number not only from cell populations but also from single cells. Our developed assay can generate data in as little as 3 h, is optimized for 96-well plates, and also allows the direct use of cell lysates without the need for DNA purification or nuclear reference genes. We show that ddMDM is able to detect differences between samples whose mtDNA copy number was close enough as to be indistinguishable by other commonly used mtDNA quantitation methods. By utilizing ddMDM, we show quantitative changes in mtDNA content per cell across a wide variety of physiological contexts including cancer progression, cell cycle progression, human T cell activation, and human aging.

4.
Acta Biomater ; 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31562987

RESUMO

Current 3D culture models to study colorectal cancer lack architectural support and signaling proteins provided by the tissue extracellular matrix (ECM) which may influence cell behavior and cancer progression. Therefore, the ability to study cancer cells in the context of a matrix that is physiologically more relevant and to understand how the ECM affects cancer progression has been understudied. To address this, we developed an ex-vivo 3D system, provided by intact wild type (WT) and colon cancer susceptible decellularized mouse colons (DMC), to support the growth of human cancer cells. DMC are free of viable cells but still contain extracellular matrix proteins including subsets of collagens. Stiffness, an important mechanical property, is also maintained in DMCs. Importantly, we observed that the DMC is permissive for cell proliferation and differentiation of a human colon cancer cell line (HT-29). Notably, the ability of cells in the WT DMC to differentiate was also greater when compared to Matrigel™, an extracellular matrix extract from a mouse tumor cell line. Additionally, we observed in invasion assays that DMC obtained from polyps from a colon cancer susceptible mouse model facilitated increased cell migration/invasion of colorectal cancer cells and immortalized non-tumor colonic epithelial cells compared to DMC from WT mice. Finally, using mass spectrometry, we identified extracellular matrix proteins that are more abundant in DMC from a colorectal cancer mouse model compared to age and sex-matched WT mice. We propose that these abundantly expressed proteins in the tumor microenvironment are potentially involved in colorectal cancer progression. STATEMENT OF SIGNIFICANCE: Decellularized matrices, when properly produced, are attractive biomaterials for tissue regeneration and replacement. We show here that the mouse decellularized matrices can also be repurposed to elucidate how the extracellular matrix influences human cell behavior and cancer progression. To do this we produce decellularized matrices, from mice colonic tissue, that have preserved tissue mechanical and structural properties. We demonstrate that the matrix better supports the differentiation of HT-29 cells, a colonic cancer cell line, compared to Matrigel™. Additionally, we show that the extracellular matrix contributes to colon cancer progression via invasion assays using extracellular matrix extracts. Finally, we use mass spectrometry to identify ECM proteins that are more abundant in colonic polyps compared to adjacent tissue regions. This model system may have therapeutic implications for colorectal cancer patients.

5.
Cell Microbiol ; : e13099, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31414579

RESUMO

Several commensal and pathogenic Gram-negative bacteria produce DNA-damaging toxins that are considered bona fide carcinogenic agents. The microbiota of colorectal cancer (CRC) patients is enriched in genotoxin-producing bacteria, but their role in the pathogenesis of CRC is poorly understood. The adenomatous polyposis coli (APC) gene is mutated in familial adenomatous polyposis and in the majority of sporadic CRCs. We investigated whether the loss of APC alters the response of colonic epithelial cells to infection by Salmonella enterica, the only genotoxin-producing bacterium associated with cancer in humans. Using 2D and organotypic 3D cultures, we found that APC deficiency was associated with sustained activation of the DNA damage response, reduced capacity to repair different types of damage, including DNA breaks and oxidative damage, and failure to induce cell cycle arrest. The reduced DNA repair capacity and inability to activate adequate checkpoint responses was associated with increased genomic instability in APC-deficient cells exposed to the genotoxic bacterium. Inhibition of the checkpoint response was dependent on activation of the phosphatidylinositol 3-kinase pathway. These findings highlight the synergistic effect of the loss of APC and infection with genotoxin-producing bacteria in promoting a microenvironment conducive to malignant transformation.

6.
Genes Dev ; 33(17-18): 1236-1251, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31416966

RESUMO

Tumors display increased uptake and processing of nutrients to fulfill the demands of rapidly proliferating cancer cells. Seminal studies have shown that the proto-oncogene MYC promotes metabolic reprogramming by altering glutamine uptake and metabolism in cancer cells. How MYC regulates the metabolism of other amino acids in cancer is not fully understood. Using high-performance liquid chromatography (HPLC)-tandem mass spectrometry (LC-MS/MS), we found that MYC increased intracellular levels of tryptophan and tryptophan metabolites in the kynurenine pathway. MYC induced the expression of the tryptophan transporters SLC7A5 and SLC1A5 and the enzyme arylformamidase (AFMID), involved in the conversion of tryptophan into kynurenine. SLC7A5, SLC1A5, and AFMID were elevated in colon cancer cells and tissues, and kynurenine was significantly greater in tumor samples than in the respective adjacent normal tissue from patients with colon cancer. Compared with normal human colonic epithelial cells, colon cancer cells were more sensitive to the depletion of tryptophan. Blocking enzymes in the kynurenine pathway caused preferential death of established colon cancer cells and transformed colonic organoids. We found that only kynurenine and no other tryptophan metabolite promotes the nuclear translocation of the transcription factor aryl hydrocarbon receptor (AHR). Blocking the interaction between AHR and kynurenine with CH223191 reduced the proliferation of colon cancer cells. Therefore, we propose that limiting cellular kynurenine or its downstream targets could present a new strategy to reduce the proliferation of MYC-dependent cancer cells.


Assuntos
Neoplasias do Colo/fisiopatologia , Cinurenina/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Triptofano/metabolismo , Sistema ASC de Transporte de Aminoácidos/genética , Antineoplásicos/farmacologia , Arilformamidase/genética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Indóis/farmacologia , Cinurenina/genética , Transportador 1 de Aminoácidos Neutros Grandes/genética , Antígenos de Histocompatibilidade Menor/genética , Oximas/farmacologia , Sulfonamidas/farmacologia
7.
Genes Dev ; 33(13-14): 814-827, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171703

RESUMO

Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. One of the hallmarks of ALT cancer is the excessively clustered telomeres in promyelocytic leukemia (PML) bodies, represented as large bright telomere foci. Here, we present a model system that generates telomere clustering in nuclear polySUMO (small ubiquitin-like modification)/polySIM (SUMO-interacting motif) condensates, analogous to PML bodies, and thus artificially engineered ALT-associated PML body (APB)-like condensates in vivo. We observed that the ALT-like phenotypes (i.e., a small fraction of heterogeneous telomere lengths and formation of C circles) are rapidly induced by introducing the APB-like condensates together with BLM through its helicase domain, accompanied by ssDNA generation and RPA accumulation at telomeres. Moreover, these events lead to mitotic DNA synthesis (MiDAS) at telomeres mediated by RAD52 through its highly conserved N-terminal domain. We propose that the clustering of large amounts of telomeres in human cancers promotes ALT that is mediated by MiDAS, analogous to Saccharomyces cerevisiae type II ALT survivors.


Assuntos
Núcleo Celular/metabolismo , DNA/biossíntese , Leucemia Promielocítica Aguda/fisiopatologia , Mitose , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , RecQ Helicases/metabolismo , Homeostase do Telômero/genética , Motivos de Aminoácidos , Linhagem Celular Tumoral , Expressão Gênica , Humanos , Leucemia Promielocítica Aguda/genética , Fenótipo , Transporte Proteico , Proteína SUMO-1/metabolismo , Telômero/genética , Telômero/metabolismo
8.
Aging Cell ; 18(4): e12979, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31152494

RESUMO

Hutchinson-Gilford progeria syndrome (HGPS) is characterized by accelerated senescence due to a de novo mutation in the LMNA gene. The mutation produces an abnormal lamin A protein called progerin that lacks the splice site necessary to remove a farnesylated domain. Subsequently, progerin accumulates in the nuclear envelope, disrupting nuclear architecture, chromatin organization, and gene expression. These alterations are often associated with rapid telomere erosion and cellular aging. Here, we further characterize the cellular and molecular abnormalities in HGPS cells and report a significant reversal of some of these abnormalities by introduction of in vitro transcribed and purified human telomerase (hTERT) mRNA. There is intra-individual heterogeneity of expression of telomere-associated proteins DNA PKcs/Ku70/Ku80, with low-expressing cells having shorter telomeres. In addition, the loss of the heterochromatin marker H3K9me3 in progeria is associated with accelerated telomere erosion. In HGPS cell lines characterized by short telomeres, transient transfections with hTERT mRNA increase telomere length, increase expression of telomere-associated proteins, increase proliferative capacity and cellular lifespan, and reverse manifestations of cellular senescence as assessed by ß-galactosidase expression and secretion of inflammatory cytokines. Unexpectedly, mRNA hTERT also improves nuclear morphology. In combination with the farnesyltransferase inhibitor (FTI) lonafarnib, hTERT mRNA promotes HGPS cell proliferation. Our findings demonstrate transient expression of human telomerase in combination with FTIs could represent an improved therapeutic approach for HGPS.

9.
J Biol Chem ; 294(30): 11579-11596, 2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31186347

RESUMO

Human telomerase maintains genome stability by adding telomeric repeats to the ends of linear chromosomes. Although previous studies have revealed profound insights into telomerase functions, the low cellular abundance of functional telomerase and the difficulties in quantifying its activity leave its thermodynamic and kinetic properties only partially characterized. Employing a stable cell line overexpressing both the human telomerase RNA component and the N-terminally biotinylated human telomerase reverse transcriptase and using a newly developed method to count individual extension products, we demonstrate here that human telomerase holoenzymes contain fast- and slow-acting catalytic sites. Surprisingly, both active sites became inactive after two consecutive rounds of catalysis, named single-run catalysis. The fast active sites turned off ∼40-fold quicker than the slow ones and exhibited higher affinities to DNA substrates. In a dimeric enzyme, the two active sites work in tandem, with the faster site functioning before the slower one, and in the monomeric enzyme, the active sites also perform single-run catalysis. Interestingly, inactive enzymes could be reactivated by intracellular telomerase-activating factors (iTAFs) from multiple cell types. We conclude that the single-run catalysis and the iTAF-triggered reactivation serve as an unprecedented control circuit for dynamic regulation of telomerase. They endow native telomerase holoenzymes with the ability to match their total number of active sites to the number of telomeres they extend. We propose that the exquisite kinetic control of telomerase activity may play important roles in both cell division and cell aging.

10.
Mol Carcinog ; 58(9): 1581-1588, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31062416

RESUMO

Engaging a telomere maintenance mechanism during DNA replication is essential for almost all advanced cancers. The conversion from normal and premalignant somatic cells to advanced malignant cells often results (85%-90%) from the reactivation of the functional ribonucleoprotein holoenzyme complex, referred to as telomerase. Modulation of the human telomerase reverse transcriptase (hTERT) appears to be rate limiting to produce functional telomerase and engage a telomere maintenance mechanism. The remaining 10% to 15% of cancers overcome progressively shortened telomeres by activating an alternative lengthening of telomeres (ALT) maintenance mechanism, through a DNA recombination pathway. Exploration into the specific mechanisms of telomere maintenance in cancer have led to the development of drugs such as Imetelstat (GRN163L), BIBR1532, 6-thio-dG, VE-822, and NVP-BEZ235 being investigated as therapeutic approaches for treating telomerase and ALT tumors. The successful use of 6-thio-dG (a nucleoside preferentially recognized by telomerase) that targets and uncaps telomeres in telomerase positive but not normal telomerase silent cells has recently shown impressive effects on multiple types of cancer. For example, 6-thio-dG overcomes therapy-resistant cancers in a fast-acting mechanism potentially providing an alternative or additional route of treatment for patients with cancer. In this perspective, we provide a synopsis of the current landscape of telomeres and telomerase processing in cancer development and how this new knowledge may improve outcomes for patients with cancer.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Telômero/efeitos dos fármacos , Humanos , Neoplasias/metabolismo , Telomerase/metabolismo , Homeostase do Telômero/efeitos dos fármacos
11.
J Med Chem ; 62(10): 5217-5241, 2019 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-31070915

RESUMO

Despite advances in targeted anticancer therapies, there are still no small-molecule-based therapies available that specifically target colorectal cancer (CRC) development and progression, the second leading cause of cancer deaths. We previously disclosed the discovery of truncating adenomatous polyposis coli (APC)-selective inhibitor 1 (TASIN-1), a small molecule that specifically targets colorectal cancer cells lines with truncating mutations in the adenomatous polyposis coli (APC) tumor suppressor gene through inhibition of cholesterol biosynthesis. Here, we report a medicinal chemistry evaluation of a collection of TASIN analogues and activity against colon cancer cell lines and an isogenic cell line pair reporting on the status of APC-dependent selectivity. A number of potent and selective analogues were identified, including compounds with good metabolic stability and pharmacokinetic properties. The compounds reported herein represent a first-in-class genotype-selective series that specifically target apc mutations present in the majority of CRC patients and serve as a translational platform toward a targeted therapy for colon cancer.

12.
Nat Rev Genet ; 20(5): 299-309, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30760854

RESUMO

Many recent advances have emerged in the telomere and telomerase fields. This Timeline article highlights the key advances that have expanded our views on the mechanistic underpinnings of telomeres and telomerase and their roles in ageing and disease. Three decades ago, the classic view was that telomeres protected the natural ends of linear chromosomes and that telomerase was a specific telomere-terminal transferase necessary for the replication of chromosome ends in single-celled organisms. While this concept is still correct, many diverse fields associated with telomeres and telomerase have substantially matured. These areas include the discovery of most of the key molecular components of telomerase, implications for limits to cellular replication, identification and characterization of human genetic disorders that result in premature telomere shortening, the concept that inhibiting telomerase might be a successful therapeutic strategy and roles for telomeres in regulating gene expression. We discuss progress in these areas and conclude with challenges and unanswered questions in the field.


Assuntos
Envelhecimento/genética , Genômica/história , Neoplasias/genética , Telomerase/genética , Telômero/química , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/metabolismo , Anormalidades Múltiplas/patologia , Envelhecimento/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA/química , DNA/genética , DNA/metabolismo , Regulação da Expressão Gênica , Genômica/métodos , História do Século XX , História do Século XXI , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Progéria/genética , Progéria/metabolismo , Progéria/patologia , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Telomerase/metabolismo , Telômero/metabolismo , Homeostase do Telômero , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo
13.
Oncogene ; 38(16): 2937-2952, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30568224

RESUMO

Alternative splicing is dysregulated in cancer cells, driving the production of isoforms that allow tumor cells to survive and continuously proliferate. Part of the reactivation of telomerase involves the splicing of hTERT transcripts to produce full-length (FL) TERT. Very few splicing factors to date have been described to interact with hTERT and promote the production of FL TERT. We recently described one such splicing factor, NOVA1, that acts as an enhancer of FL hTERT splicing, increases telomerase activity, and promotes telomere maintenance in cancer cells. NOVA1 is expressed primarily in neurons and is involved in neurogenesis. In the present studies, we describe that polypyrimidine-tract binding proteins (PTBPs), which are also typically involved in neurogenesis, are also participating in the splicing of hTERT to FL in cancer. Knockdown experiments of PTBP1 in cancer cells indicate that PTBP1 reduces hTERT FL splicing and telomerase activity. Stable knockdown of PTBP1 results in progressively shortened telomere length in H1299 and H920 lung cancer cells. RNA pulldown experiments reveal that PTBP1 interacts with hTERT pre-mRNA in a NOVA1 dependent fashion. Knockdown of PTBP1 increases the expression of PTBP2 which also interacts with NOVA1, potentially preventing the association of NOVA1 with hTERT pre-mRNA. These new data highlight that splicing in cancer cells is regulated by competition for splice sites and that combinations of splicing factors interact at cis regulatory sites on pre-mRNA transcripts. By employing hTERT as a model gene, we show the coordination of the splicing factors NOVA1 and PTBP1 in cancer by regulating telomerase that is expressed in the vast majority of cancer cell types.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas/genética , Neoplasias/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Precursores de RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Telomerase/genética , Células A549 , Processamento Alternativo/genética , Linhagem Celular , Linhagem Celular Tumoral , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Processamento de RNA/genética
14.
Life Sci Space Res (Amst) ; 19: 31-42, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30482279

RESUMO

There are considerable health risks related to ionizing and proton radiation exposure. While there is a long history of health risks associated with ionizing (photon) radiation exposure, there is a limited understanding of the long-term health risks associated with proton radiation exposure. Since proton radiation is becoming more common in cancer therapy, the long-term biological effects of proton radiation remain less well characterized in terms of radiotherapy and well as for astronauts during deep space explorations. In this study, we compared the long-term side effects of proton radiation to equivalent doses of X-rays in the initiation and progression of premalignant lesions in a lung cancer susceptible mouse model (K-rasLA1). We show proton irradiation causes more complex DNA damage that is not completely repaired resulting in increased oxidative stress in the lungs both acutely and persistently. We further observed K-rasLA1 mice irradiated with protons had an increased number and size of initiated and premalignant lesions and adenomas that were often infiltrated with inflammatory cells. Proton irradiated mice had a lower median survival and increased carcinoma incidence as compared to unirradiated controls and X-rays exposed mice. Our conclusion is that exposure to proton irradiation enhances the progression of premalignant lesions to invasive carcinomas through persistent DNA damage, chronic oxidative stress, and immunosuppression.

15.
Aging Cell ; : e12859, 2018 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-30488553

RESUMO

It is generally recognized that the function of the immune system declines with increased age and one of the major immune changes is impaired T-cell responses upon antigen presentation/stimulation. Some "high-performing" centenarians (100+ years old) are remarkably successful in escaping, or largely postponing, major age-related diseases. However, the majority of centenarians ("low-performing") have experienced these pathologies and are forced to reside in long-term nursing facilities. Previous studies have pooled all centenarians examining heterogeneous populations of resting/unstimulated peripheral blood mononuclear cells (PBMCs). T cells represent around 60% of PBMC and are in a quiescence state when unstimulated. However, upon stimulation, T cells rapidly divide and exhibit dramatic changes in gene expression. We have compared stimulated T-cell responses and identified a set of transcripts expressed in vitro that are dramatically different in high- vs. low-performing centenarians. We have also identified several other measurements that are different between high- and low-performing centenarians: (a) The amount of proliferation following in vitro stimulation is dramatically greater in high-performing centenarians compared to 67- to 83-year-old controls and low-performing centenarians; (b) telomere length is greater in the high-performing centenarians; and (c) telomerase activity following stimulation is greater in the high-performing centenarians. In addition, we have validated a number of genes whose expression is directly related to telomere length and these are potential fundamental biomarkers of aging that may influence the risk and progression of multiple aging conditions.

16.
PLoS Genet ; 14(11): e1007782, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30439955

RESUMO

Telomere-binding proteins constituting the shelterin complex have been studied primarily for telomeric functions. However, mounting evidence shows non-telomeric binding and gene regulation by shelterin factors. This raises a key question-do telomeres impact binding of shelterin proteins at distal non-telomeric sites? Here we show that binding of the telomere-repeat-binding-factor-2 (TRF2) at promoters ~60 Mb from telomeres depends on telomere length in human cells. Promoter TRF2 occupancy was depleted in cells with elongated telomeres resulting in altered TRF2-mediated transcription of distal genes. In addition, histone modifications-activation (H3K4me1 and H3K4me3) as well as silencing marks (H3K27me3)-at distal promoters were telomere length-dependent. These demonstrate that transcription, and the epigenetic state, of telomere-distal promoters can be influenced by telomere length. Molecular links between telomeres and the extra-telomeric genome, emerging from findings here, might have important implications in telomere-related physiology, particularly ageing and cancer.

17.
Genes Dev ; 32(19-20): 1303-1308, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30254109

RESUMO

MYC enhances protein synthesis by regulating genes involved in ribosome biogenesis and protein translation. Here, we show that MYC-induced protein translation is mediated by the transcription factor aryl hydrocarbon receptor (AHR), which is induced by MYC in colonic cells. AHR promotes protein synthesis by activating the transcription of genes required for ribosome biogenesis and protein translation, including OGFOD1 and NOLC1. Using surface sensing of translation (SUnSET) to measure global protein translation, we found that silencing AHR or its targets diminishes protein synthesis. Therefore, targeting AHR or its downstream pathways could provide a novel approach to limit biomass production in MYC-driven tumors.


Assuntos
Nucléolo Celular/metabolismo , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores de Hidrocarboneto Arílico/fisiologia , Animais , Linhagem Celular , Nucléolo Celular/genética , Células Cultivadas , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-myc/genética , Ratos , Receptores de Hidrocarboneto Arílico/biossíntese , Receptores de Hidrocarboneto Arílico/genética , Ativação Transcricional
18.
Nat Commun ; 9(1): 3112, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-30082712

RESUMO

Alternative splicing is dysregulated in cancer and the reactivation of telomerase involves the splicing of TERT transcripts to produce full-length (FL) TERT. Knowledge about the splicing factors that enhance or silence FL hTERT is lacking. We identified splicing factors that reduced telomerase activity and shortened telomeres using a siRNA minigene reporter screen and a lung cancer cell bioinformatics approach. A lead candidate, NOVA1, when knocked down resulted in a shift in hTERT splicing to non-catalytic isoforms, reduced telomerase activity, and progressive telomere shortening. NOVA1 knockdown also significantly altered cancer cell growth in vitro and in xenografts. Genome engineering experiments reveal that NOVA1 promotes the inclusion of exons in the reverse transcriptase domain of hTERT resulting in the production of FL hTERT transcripts. Utilizing hTERT splicing as a model splicing event in cancer may provide new insights into potentially targetable dysregulated splicing factors in cancer.

19.
Neoplasia ; 20(8): 826-837, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30015158

RESUMO

Standard and targeted cancer therapies for late-stage cancer patients almost universally fail due to tumor heterogeneity/plasticity and intrinsic or acquired drug resistance. We used the telomerase substrate nucleoside precursor, 6-thio-2'-deoxyguanosine (6-thio-dG), to target telomerase-expressing non-small cell lung cancer cells resistant to EGFR-inhibitors and commonly used chemotherapy combinations. Colony formation assays, human xenografts as well as syngeneic and genetically engineered immune competent mouse models of lung cancer were used to test the effect of 6-thio-dG on targeted therapy- and chemotherapy-resistant lung cancer human cells and mouse models. We observed that erlotinib-, paclitaxel/carboplatin-, and gemcitabine/cisplatin-resistant cells were highly sensitive to 6-thio-dG in cell culture and in mouse models. 6-thio-dG, with a known mechanism of action, is a potential novel therapeutic approach to prolong disease control of therapy-resistant lung cancer patients with minimal toxicities.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Telomerase/metabolismo , Animais , Linhagem Celular Tumoral , Desoxiguanosina/análogos & derivados , Desoxiguanosina/farmacologia , Feminino , Humanos , Camundongos , Camundongos Nus , Tionucleosídeos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
20.
Mol Cell Biol ; 38(17)2018 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-29866653

RESUMO

Adenomatous polyposis coli (APC) is a key molecule to maintain cellular homeostasis in colonic epithelium by regulating cell-cell adhesion, cell polarity, and cell migration through activating the APC-stimulated guanine nucleotide-exchange factor (Asef). The APC-activated Asef stimulates the small GTPase, which leads to decreased cell-cell adherence and cell polarity, and enhanced cell migration. In colorectal cancers, while truncated APC constitutively activates Asef and promotes cancer initiation and progression, regulation of Asef by full-length APC is still unclear. Here, we report the autoinhibition mechanism of full-length APC. We found that the armadillo repeats in full-length APC interact with the APC residues 1362 to 1540 (APC-2,3 repeats), and this interaction competes off and inhibits Asef. Deletion of APC-2,3 repeats permits Asef interactions leading to downstream signaling events, including the induction of Golgi fragmentation through the activation of the Asef-ROCK-MLC2. Truncated APC also disrupts protein trafficking and cholesterol homeostasis by inhibition of SREBP2 activity in a Golgi fragmentation-dependent manner. Our study thus uncovers the autoinhibition mechanism of full-length APC and a novel gain of function of truncated APC in regulating Golgi structure, as well as cholesterol homeostasis, which provides a potential target for pharmaceutical intervention against colon cancers.

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