RESUMO
Increased neoantigens in hypermutated cancers with DNA mismatch repair deficiency (dMMR) are proposed as the major contributor to the high objective response rate in anti-PD-1 therapy. However, the mechanism of drug resistance is not fully understood. Using tumor models defective in the MMR gene Mlh1 (dMLH1), we show that dMLH1 tumor cells accumulate cytosolic DNA and produce IFN-ß in a cGAS-STING-dependent manner, which renders dMLH1 tumors slowly progressive and highly sensitive to checkpoint blockade. In neoantigen-fixed models, dMLH1 tumors potently induce T cell priming and lose resistance to checkpoint therapy independent of tumor mutational burden. Accordingly, loss of STING or cGAS in tumor cells decreases tumor infiltration of T cells and endows resistance to checkpoint blockade. Clinically, downregulation of cGAS/STING in human dMMR cancers correlates with poor prognosis. We conclude that DNA sensing within tumor cells is essential for dMMR-triggered anti-tumor immunity. This study provides new mechanisms and biomarkers for anti-dMMR-cancer immunotherapy.
Assuntos
Inibidores de Checkpoint Imunológico/uso terapêutico , Proteínas de Membrana/genética , Proteína 1 Homóloga a MutL/deficiência , Neoplasias/genética , Nucleotidiltransferases/genética , Animais , Linhagem Celular Tumoral , Reparo de Erro de Pareamento de DNA , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interferon beta/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Transplante de Neoplasias , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Nucleotidiltransferases/metabolismo , Prognóstico , Transdução de Sinais/efeitos dos fármacosRESUMO
Cell membrane transporters facilitate the passage of nucleobases and nucleosides for nucleotide synthesis and metabolism, and are important for the delivery of nucleoside analogues used in anticancer drug therapy. Here, we investigated if cell membrane transporters are involved in the cellular uptake of the nucleoside analogue DNA damage mediator 6-thio-2'-deoxyguanosine (6-thio-dG). A large panel of non-small cell lung cancer (NSCLC) cell lines (73 of 77) were sensitive to 6-thio-dG; only four NSCLC lines were resistant to 6-thio-dG. When analyzed by microarray and RNA sequencing, the resistant NSCLC cell lines clustered together, providing a molecular signature for patients that may not respond to 6-thio-dG. Significant downregulation of solute carrier family 43 A3 (SLC43A3), an equilibrative nucleobase transporter, was identified as a candidate in this molecular resistance signature. High levels of SLC43A3 mRNA predicted sensitivity to 6-thio-dG and therefore SLC43A3 could serve as a promising biomarker for 6-thio-dG sensitivity in patients with NSCLC. SIGNIFICANCE: These findings identify a biomarker of resistance to the telomeric DNA damage mediator 6-thio-2'-deoxyguanosine.
Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Sistemas de Transporte de Aminoácidos/metabolismo , Biomarcadores Tumorais/metabolismo , Desoxiguanosina/análogos & derivados , Neoplasias Pulmonares/tratamento farmacológico , Tionucleosídeos/farmacologia , Adenocarcinoma de Pulmão/mortalidade , Adenocarcinoma de Pulmão/patologia , Sistemas de Transporte de Aminoácidos/genética , Animais , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Desoxiguanosina/farmacologia , Desoxiguanosina/uso terapêutico , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Feminino , Técnicas de Silenciamento de Genes , Humanos , Estimativa de Kaplan-Meier , Pulmão/patologia , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Camundongos , RNA Interferente Pequeno/metabolismo , Telômero/efeitos dos fármacos , Tionucleosídeos/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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 , Antígeno Neuro-Oncológico Ventral , Splicing de RNA/genéticaRESUMO
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.
Assuntos
Processamento Alternativo , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Proteínas de Ligação a RNA/genética , Telomerase/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Biologia Computacional , Deleção de Genes , Inativação Gênica , Engenharia Genética , Genoma Humano , Células HeLa , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , Mutação , Transplante de Neoplasias , Antígeno Neuro-Oncológico Ventral , Fenótipo , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Telomerase/metabolismo , Telômero/ultraestruturaRESUMO
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/métodosRESUMO
The effects of radiation in two-dimensional (2D) cell culture conditions may not recapitulate tissue responses as modeled in three-dimensional (3D) organotypic culture. In this study, we determined if the frequency of radiation-induced transformation and cancer progression differed in 3D compared to 2D culture. Telomerase immortalized human bronchial epithelial cells (HBECs) with shTP53 and mutant KRas expression were exposed to various types of radiation (gamma, (+)H, (56)Fe) in either 2D or 3D culture. After irradiation, 3D structures were dissociated and passaged as a monolayer followed by measurement of transformation, cell growth and expression analysis. Cells irradiated in 3D produced significantly fewer and smaller colonies in soft agar than their 2D-irradiated counterparts (gamma P = 0.0004; (+)H P = 0.049; (56)Fe P < 0.0001). The cell culture conditions did not affect cell killing, the ability of cells to survive in a colony formation assay, and proliferation rates after radiation-implying there was no selection against cells in or dissociated from 3D conditions. However, DNA damage repair and apoptosis markers were increased in 2D cells compared to 3D cells after radiation. Ideally, expanding the utility of 3D culture will allow for a better understanding of the biological consequences of radiation exposure.
Assuntos
Brônquios/metabolismo , Técnicas de Cultura de Células/métodos , Transformação Celular Neoplásica/efeitos da radiação , Dano ao DNA , Células Epiteliais/metabolismo , Raios gama/efeitos adversos , Apoptose/efeitos da radiação , Brônquios/patologia , Linhagem Celular Transformada , Transformação Celular Neoplásica/patologia , Células Epiteliais/patologia , Regulação da Expressão Gênica/efeitos da radiação , HumanosRESUMO
KRAS is one of the most commonly mutated oncogenes in human cancer. Mutant KRAS aberrantly regulates metabolic networks. However, the contribution of cellular metabolism to mutant KRAS tumorigenesis is not completely understood. We report that mutant KRAS regulates intracellular fatty acid metabolism through Acyl-coenzyme A (CoA) synthetase long-chain family member 3 (ACSL3), which converts fatty acids into fatty Acyl-CoA esters, the substrates for lipid synthesis and ß-oxidation. ACSL3 suppression is associated with depletion of cellular ATP and causes the death of lung cancer cells. Furthermore, mutant KRAS promotes the cellular uptake, retention, accumulation, and ß-oxidation of fatty acids in lung cancer cells in an ACSL3-dependent manner. Finally, ACSL3 is essential for mutant KRAS lung cancer tumorigenesis in vivo and is highly expressed in human lung cancer. Our data demonstrate that mutant KRAS reprograms lipid homeostasis, establishing a metabolic requirement that could be exploited for therapeutic gain.
Assuntos
Acil Coenzima A/metabolismo , Coenzima A Ligases/genética , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Carcinogênese/metabolismo , Transformação Celular Neoplásica/metabolismo , Coenzima A Ligases/metabolismo , Ácidos Graxos/metabolismo , Humanos , Ligases/metabolismo , Metabolismo dos Lipídeos/genética , Lipogênese/fisiologia , Neoplasias Pulmonares/metabolismo , Camundongos Knockout , OxirreduçãoRESUMO
DNA is organized into complex three-dimensional chromatin structures, but how this spatial organization regulates gene expression remains a central question. These DNA/chromatin looping structures can range in size from 10-20 kb (enhancers/repressors) to many megabases during intra- and inter-chromosomal interactions. Recently, the influence of telomere length on chromatin organization prior to senescence has revealed the existence of long-distance chromatin loops that dictate the expression of genes located up to 10 Mb from the telomeres (Telomere Position Effect-Over Long Distances [TPE-OLD]). Here, we demonstrate the existence of a telomere loop at the 4q35 locus involving the sorbin and SH3 domain-containing protein 2 gene, SORBS2, a skeletal muscle protein using a modification of the chromosome conformation capture method. The loop reveals a cis-acting mechanism modifying SORBS2 transcription. The expression of this gene is altered by TPE-OLD in myoblasts from patients affected with the age-associated genetic disease, facioscapulohumeral muscular dystrophy (FSHD1A, MIM 158900). SORBS2 is expressed in FSHD myoblasts with short telomeres, while not detectable in FSHD myoblasts with long telomeres or in healthy myoblasts regardless of telomere length. This indicates that TPE-OLD may modify the regulation of the 4q35 locus in a pathogenic context. Upon differentiation, both FSHD and healthy myotubes express SORBS2, suggesting that SORBS2 is normally up-regulated by maturation/differentiation of skeletal muscle and is misregulated by TPE-OLD-dependent variegation in FSHD myoblasts. These findings provide additional insights for the complexity and age-related symptoms of FSHD.
Assuntos
Proteínas de Homeodomínio/genética , Células Musculares/metabolismo , Distrofia Muscular Facioescapuloumeral/genética , Encurtamento do Telômero , Telômero/genética , Ativação Transcricional , Proteínas Adaptadoras de Transdução de Sinal , Biópsia , Deleção Cromossômica , Cromossomos Humanos Par 4 , Metilação de DNA , Epistasia Genética , Regulação da Expressão Gênica , Loci Gênicos , Proteínas de Homeodomínio/metabolismo , Humanos , Hibridização in Situ Fluorescente , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Facioescapuloumeral/metabolismo , Distrofia Muscular Facioescapuloumeral/patologia , Proteína MyoD/genética , Proteína MyoD/metabolismo , Mioblastos , Proteínas de Ligação a RNARESUMO
While global chromatin conformation studies are emerging, very little is known about the chromatin conformation of human telomeres. Most studies have focused on the role of telomeres as a tumor suppressor mechanism. Here we describe how telomere length regulates gene expression long before telomeres become short enough to produce a DNA damage response (senescence). We directly mapped the interactions adjacent to specific telomere ends using a Hi-C (chromosome capture followed by high-throughput sequencing) technique modified to enrich for specific genomic regions. We demonstrate that chromosome looping brings the telomere close to genes up to 10 Mb away from the telomere when telomeres are long and that the same loci become separated when telomeres are short. Furthermore, expression array analysis reveals that many loci, including noncoding RNAs, may be regulated by telomere length. We report three genes (ISG15 [interferon-stimulated gene 15 kd], DSP [Desmoplakin], and C1S [complement component 1s subcomplement]) located at three different subtelomeric ends (1p, 6p, and 12p) whose expressions are altered with telomere length. Additionally, we confirmed by in situ analysis (3D-FISH [three-dimensional fluorescence in situ hybridization]) that chromosomal looping occurs between the loci of those genes and their respective telomere ends. We term this process TPE-OLD for "telomere position effect over long distances." Our results suggest a potential novel mechanism for how telomere shortening could contribute to aging and disease initiation/progression in human cells long before the induction of a critical DNA damage response.
Assuntos
Regulação da Expressão Gênica , Encurtamento do Telômero/genética , Telômero/genética , Telômero/metabolismo , Células Cultivadas , Cromatina/metabolismo , Perfilação da Expressão Gênica , Humanos , Mioblastos/citologiaRESUMO
Despite evidence that kinesin family member 14 (KIF14) can serve as a prognostic biomarker in various solid tumors, how it contributes to tumorigenesis remains unclear. We observed that experimental decrease in KIF14 expression increases docetaxel chemosensitivity in estrogen receptor-negative/progesterone receptor-negative/human epidermal growth factor receptor 2-negative, "triple-negative" breast cancers (TNBC). To investigate the oncogenic role of KIF14, we used noncancerous human mammary epithelial cells and ectopically expressed KIF14 and found increased proliferative capacity, increased anchorage-independent grown in vitro, and increased resistance to docetaxel but not to doxorubicin, carboplatin, or gemcitabine. Seventeen benign breast biopsies of BRCA1 or BRCA2 mutation carriers showed increased KIF14 mRNA expression by fluorescence in situ hybridization compared to controls with no known mutations in BRCA1 or BRCA2, suggesting increased KIF14 expression as a biomarker of high-risk breast tissue. Evaluation of 34 cases of locally advanced TNBC showed that KIF14 expression significantly correlates with chemotherapy-resistant breast cancer. KIF14 knockdown also correlates with decreased AKT phosphorylation and activity. Live-cell imaging confirmed an insulin-induced temporal colocalization of KIF14 and AKT at the plasma membrane, suggesting a potential role of KIF14 in promoting activation of AKT. An experimental small-molecule inhibitor of KIF14 was then used to evaluate the potential anticancer benefits of downregulating KIF14 activity. Inhibition of KIF14 shows a chemosensitizing effect and correlates with decreasing activation of AKT. Together, these findings show an early and critical role for KIF14 in the tumorigenic potential of TNBC, and therapeutic targeting of KIF14 is feasible and effective for TNBC.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Cinesinas/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Proteína BRCA1/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Heterozigoto , Humanos , Cinesinas/genética , Terapia Neoadjuvante , Proteínas Oncogênicas/genética , Fosforilação , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
INTRODUCTION: Metastasis is the main cause of breast cancer morbidity and mortality. Processes that allow for tumor cell migration and invasion are important therapeutic targets. Here we demonstrate that receptor-interacting protein kinase 2 (RIP2), a kinase known to be involved in inflammatory processes, also has novel roles in cancer cell migration and invasion. METHODS: A total of six breast cancer expression databases, including The Cancer Genome Atlas, were assessed for RIP2 expression among various clinical subtypes and its role as a prognostic biomarker. mRNA fluorescence in situ hybridization (FISH) for RIP2 was performed on 17 stage III breast cancers to determine if there was a correlation between RIP2 expression and lymph node involvement. RNA-interference was used to knock-down RIP2 expression in MDA-MB-231, Htb126, SUM149PT, MCF7, T47D, and HCC1428 cells. Cell migration and invasion were measured in vitro by scratch/wound healing and transwell migration assays. A xenograft mouse model was used to assess tumor growth and chemosensitivity to docetaxel in vivo in MDA-MB-231 cells with and without RIP2 small hairpin RNA knockdown. Western blot and immunofluorescence imaging were used to evaluate protein expressions. RESULTS: Interrogation of expression databases showed that RIP2 expression is significantly over-expressed in triple-negative breast cancers (TNBC: estrogen-receptor (ER) negative, progesterone-receptor (PR) negative, Her2/neu- (Her2) negative), compared to other clinical subtypes. High RIP2 expression correlates with worse progression-free survival using a combined breast cancer expression array dataset consisting of 946 patients. Multivariate analysis shows RIP2 as an independent prognostic biomarker. Knock-down of RIP2 significantly decreases migration in both scratch/wound healing and transwell migration assays in MDA-MB-231, Htb126, SUM149PT, MCF7, and T47D cells and is correlated with decreased Nuclear Factor-kappaB and c-Jun N-terminal kinase (JNK) activation. Finally, RIP2 knock-down leads to increased sensitivity to docetaxel and decreased tumor mass and lung metastases in a xenograft mouse model. CONCLUSION: These results highlight RIP2 as a pro-metastasis kinase in patients with advanced breast cancer. These results also illustrate a novel role for this kinase in addition to its known role in inflammation, and suggest that targeting RIP2 may improve outcomes in advanced breast cancer patients, in which it is overexpressed.
Assuntos
Movimento Celular/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/genética , Antineoplásicos/farmacologia , Western Blotting , Linhagem Celular Tumoral , Docetaxel , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Células MCF-7 , Microscopia de Fluorescência , Invasividade Neoplásica , Prognóstico , Interferência de RNA , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Taxoides/farmacologia , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Carcinogenesis is an adaptive process between nascent tumor cells and their microenvironment, including the modification of inflammatory responses from antitumorigenic to protumorigenic. Radiation exposure can stimulate inflammatory responses that inhibit or promote carcinogenesis. The purpose of this study is to determine the impact of radiation exposure on lung cancer progression in vivo and assess the relevance of this knowledge to human carcinogenesis. EXPERIMENTAL DESIGN: K-ras(LA1) mice were irradiated with various doses and dose regimens and then monitored until death. Microarray analyses were performed using Illumina BeadChips on whole lung tissue 70 days after irradiation with a fractionated or acute dose of radiation and compared with age-matched unirradiated controls. Unique group classifiers were derived by comparative genomic analysis of three experimental cohorts. Survival analyses were performed using principal component analysis and k-means clustering on three lung adenocarcinoma, three breast adenocarcinoma, and two lung squamous carcinoma annotated microarray datasets. RESULTS: Radiation exposure accelerates lung cancer progression in the K-ras(LA1) lung cancer mouse model with dose fractionation being more permissive for cancer progression. A nonrandom inflammatory signature associated with this progression was elicited from whole lung tissue containing only benign lesions and predicts human lung and breast cancer patient survival across multiple datasets. Immunohistochemical analyses suggest that tumor cells drive predictive signature. CONCLUSIONS: These results demonstrate that radiation exposure can cooperate with benign lesions in a transgenic model of cancer by affecting inflammatory pathways, and that clinically relevant similarities exist between human lung and breast carcinogenesis.
Assuntos
Carcinoma/patologia , Transformação Celular Neoplásica/efeitos da radiação , Neoplasias Pulmonares/patologia , Neoplasias Induzidas por Radiação/patologia , Lesões Experimentais por Radiação/patologia , Animais , Western Blotting , Neoplasias da Mama/patologia , Neoplasias da Mama/radioterapia , Carcinoma/radioterapia , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/radioterapia , Masculino , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Componente PrincipalRESUMO
PURPOSE: To identify biomarkers within the breast cancer genome that may predict chemosensitivity in breast cancer. EXPERIMENTAL DESIGN: We conducted an RNA interference (RNAi) screen within the breast cancer genome for genes whose loss-of-function enhanced docetaxel chemosensitivity in an estrogen receptor-negative, progesterone receptor-negative, and Her2-negative (ER-, PR-, and Her2-, respectively) breast cancer cell line, MDA-MB-231. Top candidates were tested for their ability to modulate chemosensitivity in 8 breast cancer cell lines and to show in vivo chemosensitivity in a mouse xenograft model. RESULTS: From ranking chemosensitivity of 328 short hairpin RNA (shRNA) MDA-MB-231 cell lines (targeting 133 genes with known somatic mutations in breast cancer), we focused on the top two genes, kinesin family member 14 (KIF14) and talin 1 (TLN1). KIF14 and TLN1 loss-of-function significantly enhanced chemosensitivity in four triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, HCC38, HCC1937, and Hs478T) but not in three hormone receptor-positive cell lines (MCF7, T47D, and HCC1428) or normal human mammary epithelial cells (HMEC). Decreased expression of KIF14, but not TLN1, also enhanced docetaxel sensitivity in a Her2-amplified breast cancer cell line, SUM190PT. Higher KIF14 and TLN1 expressions are found in TNBCs compared with the other clinical subtypes. Mammary fat pad xenografts of KIF14- and TLN1-deficient MDA-MB-231 cells revealed reduced tumor mass compared with control MDA-MB-231 cells after chemotherapy. KIF14 expression is also prognostic of relapse-free and overall survival in representative breast cancer expression arrays. CONCLUSION: KIF14 and TLN1 are modulators of response to docetaxel and potential therapeutic targets in TNBC.
Assuntos
Neoplasias da Mama/genética , Cinesinas/genética , Proteínas Oncogênicas/genética , Interferência de RNA , Talina/genética , Taxoides/farmacologia , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Docetaxel , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Estudo de Associação Genômica Ampla/métodos , Humanos , Estimativa de Kaplan-Meier , Cinesinas/metabolismo , Células MCF-7 , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Oncogênicas/metabolismo , Prognóstico , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Talina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
While the adult murine lung utilizes multiple compartmentally restricted progenitor cells during homeostasis and repair, much less is known about the progenitor cells from the human lung. Translating the murine stem cell model to humans is hindered by anatomical differences between species. Here we show that human bronchial epithelial cells (HBECs) display characteristics of multipotent stem cells of the lung. These HBECs express markers indicative of several epithelial types of the adult lung when experimentally tested in cell culture. When cultured in three different three-dimensional (3D) systems, subtle changes in the microenvironment result in unique responses including the ability of HBECs to differentiate into multiple central and peripheral lung cell types. These new findings indicate that the adult human lung contains a multipotent progenitor cell whose differentiation potential is primarily dictated by the microenvironment. The HBEC system is not only important in understanding mechanisms for specific cell lineage differentiation, but also for examining changes that correlate with human lung diseases including lung cancer.
Assuntos
Brônquios/citologia , Células Epiteliais/citologia , Células-Tronco Multipotentes/citologia , Biomarcadores/metabolismo , Diferenciação Celular/genética , Linhagem Celular Transformada , Células Cultivadas , Quinase 4 Dependente de Ciclina/metabolismo , Células Epiteliais/enzimologia , Células Epiteliais/ultraestrutura , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Modelos Biológicos , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/metabolismo , Telomerase/metabolismo , Técnicas de Cultura de TecidosRESUMO
Chromosomal instability leading to aneuploidy occurs in most sporadic colorectal cancers (CRCs) and is believed to be an early driving force in disease progression. Despite this observation, the cellular advantages conferred by these cytogenetic alterations are poorly understood. Here, we provide evidence that serum-free passage of originally diploid, immortalized human colonic epithelial cells (HCECs) gave rise to the acquisition of trisomy 7 (+7), an aneuploidy detected in more than 40% of colorectal adenomas. These cells remain diploid under long-term growth in 2% serum conditions. Analysis by GTG banding and fluorescent in situ hybridization detected no rare preexisting +7 cell in the original population, suggesting a conversion of diploid cells to an aneuploid state. The acquisition of +7 also precedes loss or truncation of the adenomatosis polyposis coli gene as both diploid and +7 cells express full-length, functional protein. Coculturing of fluorescent-labeled cells demonstrate that +7 HCECs have a growth advantage over diploid cells in serum-free conditions. Defects in cell migration and aberrant regulation of the epidermal growth factor receptor, located on chromosome 7p, are also detected in +7 HCECs. Interestingly, knockdown of TP53 and expression of K-Ras(V12) in +7 HCECs resulted in the emergence of trisomy 20, another nonrandom aneuploidy observed in â¼85% of CRC. In summary, we describe isogenic colonic epithelial cells that represent cytogenetic changes occurring frequently in sporadic CRC. The emergence and characterization of trisomy 7 and 20 demonstrate that these HCECs may serve as unique human cell-based models to examine the effects of chromosomal instability in CRC progression.
Assuntos
Cromossomos Humanos Par 7 , Colo/patologia , Diploide , Células Epiteliais/patologia , Trissomia/patologia , Aneuploidia , Carcinoma/genética , Carcinoma/patologia , Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Células Cultivadas , Instabilidade Cromossômica , Cromossomos Humanos Par 20/genética , Colo/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Meios de Cultura Livres de Soro/farmacologia , Análise Citogenética , Células Epiteliais/metabolismo , Deleção de Genes , Genes APC/fisiologia , Humanos , Mosaicismo , Fatores de Tempo , Trissomia/genéticaRESUMO
Radiation-induced carcinogenesis is a major concern both for astronauts on long-term space missions and for cancer patients being treated with therapeutic radiation. Exposure to radiation induces oxidative stress and chronic inflammation, which are critical initiators and promoters of carcinogenesis. Many studies have demonstrated that non-steroidal anti-inflammatory drugs and antioxidants can reduce the risk of radiation-induced cancer. In this study, we found that a synthetic triterpenoid, CDDO-Me (bardoxolone methyl), was able to protect human colon epithelial cells (HCECs) against radiation-induced transformation. HCECs that were immortalized by ectopic expression of hTERT and cdk4 and exhibit trisomy for chromosome 7 (a non-random chromosome change that occurs in 37% of premalignant colon adenomas) can be transformed experimentally with one combined exposure to 2 Gy of protons at 1 GeV/nucleon followed 24 h later by 50 cGy of (56)Fe ions at 1 GeV/nucleon. Transformed cells showed an increase in proliferation rate and in both anchorage-dependent and independent colony formation ability. A spectrum of chromosome aberrations was observed in transformed cells, with 40% showing loss of 17p (e.g. loss of one copy of p53). Pretreatment of cells with pharmacological doses of CDDO-Me, which has been shown to induce antioxidative as well as anti-inflammatory responses, prevented the heavy-ion-induced increase in proliferation rate and anchorage-dependent and independent colony formation efficiencies. Taken together, these results demonstrate that experimentally immortalized human colon epithelial cells with a non-random chromosome 7 trisomy are valuable premalignant cellular reagents that can be used to study radiation-induced colorectal carcinogenesis. The utility of premalignant HCECs to test novel compounds such as CDDO-Me that can be used to protect against radiation-induced neoplastic transformation is also demonstrated.