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1.
J Biol Chem ; 300(6): 107380, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38762178

RESUMO

Cancer testis antigens (CTAs) are a collection of proteins whose expression is normally restricted to the gamete but abnormally activated in a wide variety of tumors. The CTA, Testis-specific serine kinase 6 (TSSK6), is essential for male fertility in mice. The functional relevance of TSSK6 to cancer, if any, has not previously been investigated. Here we find that TSSK6 is frequently anomalously expressed in colorectal cancer and patients with elevated TSSK6 expression have reduced relapse-free survival. Depletion of TSSK6 from colorectal cancer cells attenuates anchorage-independent growth, invasion, and growth in vivo. Conversely, overexpression of TSSK6 enhances anchorage independence and invasion in vitro as well as in vivo tumor growth. Notably, ectopic expression of TSSK6 in semi-transformed human colonic epithelial cells is sufficient to confer anchorage independence and enhance invasion. In somatic cells, TSSK6 co-localizes with and enhances the formation of paxillin and tensin-positive foci at the cell periphery, suggesting a function in focal adhesion formation. Importantly, TSSK6 kinase activity is essential to induce these tumorigenic behaviors. Our findings establish that TSSK6 exhibits oncogenic activity when abnormally expressed in colorectal cancer cells. Thus, TSSK6 is a previously unrecognized intervention target for therapy, which could exhibit an exceptionally broad therapeutic window.


Assuntos
Neoplasias Colorretais , Regulação Neoplásica da Expressão Gênica , Proteínas Serina-Treonina Quinases , Animais , Humanos , Masculino , Camundongos , Carcinogênese/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Neoplasias Colorretais/genética , Adesões Focais/metabolismo , Adesões Focais/genética , Invasividade Neoplásica , Paxilina/metabolismo , Paxilina/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Tensinas/metabolismo , Tensinas/genética
2.
J Biol Chem ; 299(11): 105348, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37838177

RESUMO

Tumors anomalously induce the expression of meiotic genes, which are otherwise restricted only to developing gametes. If and how these aberrantly expressed meiotic proteins influence DNA metabolism is not clear, but could have important implications for how tumors acquire and mitigate genomic instability. HORMAD1 is a highly conserved meiotic protein that is frequently expressed in lung adenocarincoma where its expression correlates with reduced patient survival and increased mutation burden. Here, we find that HORMAD1 associates with the replisome and is critical for protecting stalled DNA replication forks. Loss of HORMAD1 leads to nascent DNA strand degradation, an event which is mediated by the MRE11-DNA2-BLM pathway. We find that these phenotypes are due to limited RAD51 loading onto stalled replication forks in the absence of HORMAD1. Ultimately, loss of HORMAD1 leads to increased DNA breaks and chromosomal defects, which is exacerbated dramatically by induction of replication stress. Tumor cells proliferate despite encountering chronic replication stress, placing them on the precipice of catastrophic genomic damage. Our data support the hypothesis that the aberrant expression of HORMAD1 is engaged to attenuate the accumulation of excessive DNA damage due to chronic replication stress, which may otherwise lead to accumulation of toxic levels of genomic instability.


Assuntos
Proteínas de Ciclo Celular , Replicação do DNA , Neoplasias , Humanos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica , Neoplasias/genética
3.
Mol Cell ; 54(5): 805-19, 2014 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-24793696

RESUMO

The Cullin 9 (CUL9) gene encodes a putative E3 ligase that localizes in the cytoplasm. Cul9 null mice develop spontaneous tumors in multiple organs; however, both the cellular and the molecular mechanisms of CUL9 in tumor suppression are currently unknown. We show here that deletion of Cul9 leads to abnormal nuclear morphology, increased DNA damage, and aneuploidy. CUL9 knockdown rescues the microtubule and mitosis defects in cells depleted for CUL7 or OBSL1, two genes that are mutated in a mutually exclusive manner in 3M growth retardation syndrome and function in microtubule dynamics. CUL9 promotes the ubiquitylation and degradation of survivin and is inhibited by CUL7. Depletion of CUL7 decreases survivin level, and overexpression of survivin rescues the defects caused by CUL7 depletion. We propose a 3M-CUL9-survivin pathway in maintaining microtubule and genome integrity, normal development, and tumor suppression.


Assuntos
Proteínas Culina/fisiologia , Instabilidade Genômica , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitinação , Aneuploidia , Animais , Morte Celular , Proteínas Culina/genética , Proteínas Culina/metabolismo , Técnicas de Silenciamento de Genes , Genes Supressores de Tumor , Células HCT116 , Humanos , Fígado/patologia , Camundongos , Camundongos Knockout , Complexos Multiproteicos/fisiologia , Poliploidia , Multimerização Proteica , Survivina
4.
Mol Cell ; 54(5): 791-804, 2014 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-24793695

RESUMO

CUL7, OBSL1, and CCDC8 genes are mutated in a mutually exclusive manner in 3M and other growth retardation syndromes. The mechanism underlying the function of the three 3M genes in development is not known. We found that OBSL1 and CCDC8 form a complex with CUL7 and regulate the level and centrosomal localization of CUL7, respectively. CUL7 depletion results in altered microtubule dynamics, prometaphase arrest, tetraploidy, and mitotic cell death. These defects are recaptured in CUL7 mutated 3M cells and can be rescued by wild-type, but not by 3M patient-derived CUL7 mutants. Depletion of either OBSL1 or CCDC8 results in defects and sensitizes cells to microtubule damage similarly to loss of CUL7 function. Microtubule damage reduces the level of CCDC8 that is required for the centrosomal localization of CUL7. We propose that CUL7, OBSL1, and CCDC8 proteins form a 3M complex that functions in maintaining microtubule and genome integrity and normal development.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas Culina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Instabilidade Genômica , Microtúbulos/metabolismo , Linhagem Celular Tumoral , Centrossomo/metabolismo , Proteínas Culina/genética , Nanismo/genética , Proteínas F-Box/metabolismo , Genoma Humano , Células HEK293 , Humanos , Hipotonia Muscular/genética , Mutação de Sentido Incorreto , Transporte Proteico , Fuso Acromático/metabolismo , Coluna Vertebral/anormalidades
5.
Proc Natl Acad Sci U S A ; 113(50): 14342-14347, 2016 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-27911840

RESUMO

The with-no-lysine (K) (WNK) kinases are an atypical family of protein kinases that regulate ion transport across cell membranes. Mutations that result in their overexpression cause hypertension-related disorders in humans. Of the four mammalian WNKs, only WNK1 is expressed throughout the body. We report that WNK1 inhibits autophagy, an intracellular degradation pathway implicated in several human diseases. Using small-interfering RNA-mediated WNK1 knockdown, we show autophagosome formation and autophagic flux are accelerated. In cells with reduced WNK1, basal and starvation-induced autophagy is increased. We also show that depletion of WNK1 stimulates focal class III phosphatidylinositol 3-kinase complex (PI3KC3) activity, which is required to induce autophagy. Depletion of WNK1 increases the expression of the PI3KC3 upstream regulator unc-51-like kinase 1 (ULK1), its phosphorylation, and activation of the kinase upstream of ULK1, the AMP-activated protein kinase. In addition, we show that the N-terminal region of WNK1 binds to the UV radiation resistance-associated gene (UVRAG) in vitro and WNK1 partially colocalizes with UVRAG, a component of a PI3KC3 complex. This colocalization decreases upon starvation of cells. Depletion of the SPS/STE20-related proline-alanine-rich kinase, a WNK1-activated enzyme, also induces autophagy in nutrient-replete or -starved conditions, but depletion of the related kinase and WNK1 substrate, oxidative stress responsive 1, does not. These results indicate that WNK1 inhibits autophagy by multiple mechanisms.


Assuntos
Autofagia/fisiologia , Proteína Quinase 1 Deficiente de Lisina WNK/fisiologia , Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Linhagem Celular , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Biológicos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNK/antagonistas & inibidores , Proteína Quinase 1 Deficiente de Lisina WNK/genética
6.
Annu Rev Pharmacol Toxicol ; 54: 251-72, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24160706

RESUMO

Tumor cells frequently exhibit widespread epigenetic aberrations that significantly alter the repertoire of expressed proteins. In particular, it has been known for nearly 25 years that tumors frequently reactivate genes whose expression is typically restricted to germ cells. These gene products are classified as cancer/testis antigens (CTAs) owing to their biased expression pattern and their immunogenicity in cancer patients. While these genes have been pursued as targets for anticancer vaccines, whether these reactivated testis proteins have roles in supporting tumorigenic features is less studied. Recent evidence now indicates that these proteins can be directly employed by the tumor cell regulatory environment to support cell-autonomous behaviors. Here, we review the history of the CTA field and present recent findings indicating that CTAs can play functional roles in supporting tumorigenesis.


Assuntos
Antígenos de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Testiculares/genética , Testículo/imunologia , Antígenos de Neoplasias/imunologia , Humanos , Masculino , Neoplasias Testiculares/imunologia , Ativação Transcricional
7.
Nanomedicine ; 10(2): 321-8, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23916886

RESUMO

Current preclinical evaluations of nanoparticle taxanes have focused on the effect of nanoparticle size and shape on the efficacy and toxicity. It is generally assumed that nanoparticle therapeutics have the same cellular response on tumor and normal cells as their small molecule counterparts. Here, we show that nanoparticle taxanes can mediate cellular effects distinct from that of small molecule taxanes at the sub-therapeutic dose range. Cells that are exposed to two polymeric nanoparticle formulations of docetaxel were found to undergo a different cell cycle and cell fate than those of cells that were exposed to small molecule docetaxel. Our results suggest that nanoparticle formulation of therapeutics can affect the therapeutic effect of its cargo. FROM THE CLINICAL EDITOR: This study investigates the differences between subtherapeutic doses of docetaxel applied as small molecules vs. nanoparticle formulations, demonstrating differential effects on the cell cycle and overall cell fate. The study suggests that the carrier may change the therapeutic effects of its cargo, which has important implications on future research.


Assuntos
Antineoplásicos/química , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Taxoides/química , Linhagem Celular Tumoral , Docetaxel , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/química , Humanos , Micelas , Nanomedicina , Tamanho da Partícula , Polímeros/química , Taxoides/administração & dosagem
8.
Cancer Res ; 84(5): 675-687, 2024 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-38190717

RESUMO

Therapy resistance and metastatic progression are primary causes of cancer-related mortality. Disseminated tumor cells possess adaptive traits that enable them to reprogram their metabolism, maintain stemness, and resist cell death, facilitating their persistence to drive recurrence. The survival of disseminated tumor cells also depends on their ability to modulate replication stress in response to therapy while colonizing inhospitable microenvironments. In this study, we discovered that the nuclear translocation of AXL, a TAM receptor tyrosine kinase, and its interaction with WRNIP1, a DNA replication stress response factor, promotes the survival of HER2+ breast cancer cells that are resistant to HER2-targeted therapy and metastasize to the brain. In preclinical models, knocking down or pharmacologically inhibiting AXL or WRNIP1 attenuated protection of stalled replication forks. Furthermore, deficiency or inhibition of AXL and WRNIP1 also prolonged metastatic latency and delayed relapse. Together, these findings suggest that targeting the replication stress response, which is a shared adaptive mechanism in therapy-resistant and metastasis-initiating cells, could reduce metachronous metastasis and enhance the response to standard-of-care therapies. SIGNIFICANCE: Nuclear AXL and WRNIP1 interact and mediate replication stress response, promote therapy resistance, and support metastatic progression, indicating that targeting the AXL/WRNIP1 axis is a potentially viable therapeutic strategy for breast cancer.


Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Receptor Tirosina Quinase Axl , Proteínas Proto-Oncogênicas/metabolismo , Recidiva Local de Neoplasia , Receptores Proteína Tirosina Quinases/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular Tumoral , Microambiente Tumoral , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Ligação a DNA/metabolismo
9.
Nature ; 446(7137): 815-9, 2007 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-17429401

RESUMO

Abundant evidence suggests that a unifying principle governing the molecular pathology of cancer is the co-dependent aberrant regulation of core machinery driving proliferation and suppressing apoptosis. Anomalous proteins engaged in support of this tumorigenic regulatory environment most probably represent optimal intervention targets in a heterogeneous population of cancer cells. The advent of RNA-mediated interference (RNAi)-based functional genomics provides the opportunity to derive unbiased comprehensive collections of validated gene targets supporting critical biological systems outside the framework of preconceived notions of mechanistic relationships. We have combined a high-throughput cell-based one-well/one-gene screening platform with a genome-wide synthetic library of chemically synthesized small interfering RNAs for systematic interrogation of the molecular underpinnings of cancer cell chemoresponsiveness. NCI-H1155, a human non-small-cell lung cancer line, was employed in a paclitaxel-dependent synthetic lethal screen designed to identify gene targets that specifically reduce cell viability in the presence of otherwise sublethal concentrations of paclitaxel. Using a stringent objective statistical algorithm to reduce false discovery rates below 5%, we isolated a panel of 87 genes that represent major focal points of the autonomous response of cancer cells to the abrogation of microtubule dynamics. Here we show that several of these targets sensitize lung cancer cells to paclitaxel concentrations 1,000-fold lower than otherwise required for a significant response, and we identify mechanistic relationships between cancer-associated aberrant gene expression programmes and the basic cellular machinery required for robust mitotic progression.


Assuntos
Genes Letais/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Paclitaxel/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Análise Mutacional de DNA , Relação Dose-Resposta a Droga , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/tratamento farmacológico , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Mitose/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismo , Fuso Acromático/patologia
10.
bioRxiv ; 2023 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-36778501

RESUMO

Tumors frequently activate the expression of genes that are only otherwise required for meiosis. HORMAD1, which is essential for meiotic recombination in multiple species, is expressed in over 50% of human lung adenocarcinoma cells (LUAD). We previously found that HORMAD1 promotes DNA double strand break (DSB) repair in LUAD. Here, we report that HORMAD1 takes on an additional role in protecting genomic integrity. Specifically, we find HORMAD1 is critical for protecting stalled DNA replication forks in LUAD. Loss of HORMAD1 leads to nascent DNA degradation, an event which is mediated by the MRE11-DNA2-BLM pathway. Moreover, following exogenous induction of DNA replication stress, HORMAD1 deleted cells accumulate single stranded DNA (ssDNA). We find that these phenotypes are the result of a lack of RAD51 and BRCA2 loading onto stalled replication forks. Ultimately, loss of HORMAD1 leads to increased DSBs and chromosomal aberrations in response to replication stress. Collectively, our data support a model where HORMAD1 expression is selected to mitigate DNA replication stress, which would otherwise induce deleterious genomic instability.

11.
Elife ; 92020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32990599

RESUMO

Cancer testis antigens (CTAs) are proteins whose expression is normally restricted to the testis but anomalously activated in human cancer. In sperm, a number of CTAs support energy generation, however, whether they contribute to tumor metabolism is not understood. We describe human COX6B2, a component of cytochrome c oxidase (complex IV). COX6B2 is expressed in human lung adenocarcinoma (LUAD) and expression correlates with reduced survival time. COX6B2, but not its somatic isoform COX6B1, enhances activity of complex IV, increasing oxidative phosphorylation (OXPHOS) and NAD+ generation. Consequently, COX6B2-expressing cancer cells display a proliferative advantage, particularly in low oxygen. Conversely, depletion of COX6B2 attenuates OXPHOS and collapses mitochondrial membrane potential leading to cell death or senescence. COX6B2 is both necessary and sufficient for growth of human tumor xenografts in mice. Our findings reveal a previously unappreciated, tumor-specific metabolic pathway hijacked from one of the most ATP-intensive processes in the animal kingdom: sperm motility.


Assuntos
Adenocarcinoma de Pulmão/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Neoplasias Pulmonares/genética , Fosforilação Oxidativa , Animais , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Nus
12.
Elife ; 92020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32515734

RESUMO

Cancer/testis (CT) antigens are proteins whose expression is normally restricted to germ cells yet aberrantly activated in tumors, where their functions remain relatively cryptic. Here we report that ZNF165, a CT antigen frequently expressed in triple-negative breast cancer (TNBC), associates with SMAD3 to modulate transcription of transforming growth factor ß (TGFß)-dependent genes and thereby promote growth and survival of human TNBC cells. In addition, we identify the KRAB zinc finger protein, ZNF446, and its associated tripartite motif protein, TRIM27, as obligate components of the ZNF165-SMAD3 complex that also support tumor cell viability. Importantly, we find that TRIM27 alone is necessary for ZNF165 transcriptional activity and is required for TNBC tumor growth in vivo using an orthotopic xenograft model in immunocompromised mice. Our findings indicate that aberrant expression of a testis-specific transcription factor is sufficient to co-opt somatic transcriptional machinery to drive a pro-tumorigenic gene expression program in TNBC.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteína Smad3/metabolismo , Testículo/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Transdução de Sinais , Proteína Smad3/genética , Neoplasias de Mama Triplo Negativas/genética
13.
Mol Cell Biol ; 39(14)2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31036566

RESUMO

Ewing sarcoma is characterized by a pathognomonic chromosomal translocation that generates the EWSR1-FLI1 chimeric transcription factor. The transcriptional targets of EWSR1-FLI1 that are essential for tumorigenicity are incompletely defined. Here, we found that EWSR1-FLI1 modulates the expression of cancer/testis (CT) antigen genes, whose expression is biased to the testes but is also activated in cancer. Among these CT antigens, fetal and adult testis expressed 1 (FATE1) is most robustly induced. EWSR1-FLI1 associates with the GGAA repeats in the proximal promoter of FATE1, which exhibits accessible chromatin exclusively in mesenchymal progenitor cells (MPCs) and Ewing sarcoma cells. Expression of EWSR1-FLI1 in non-Ewing sarcoma cells and in MPCs enhances FATE1 mRNA and protein expression. Conversely, depletion of EWSR1-FLI1 in Ewing sarcoma cells leads to a loss of FATE1 expression. Importantly, we found that FATE1 is required for survival and anchorage-independent growth in Ewing sarcoma cells via attenuating the accumulation of BNIP3L, a BH3-only protein that is toxic when stabilized. This action appears to be mediated by the E3 ligase RNF183. We propose that engaging FATE1 function can permit the bypass of cell death mechanisms that would otherwise inhibit tumor progression.


Assuntos
Neoplasias Ósseas/genética , Proteínas de Ligação a DNA/genética , Proteínas de Fusão Oncogênica/genética , Sarcoma de Ewing/genética , Fatores de Transcrição/genética , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HeLa , Células Hep G2 , Humanos , Proteínas de Membrana/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Sarcoma de Ewing/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
14.
Trends Cancer ; 4(10): 701-712, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30292353

RESUMO

Tumors of nearly every origin activate the expression of genes normally restricted to gametogenic cells. These genes encode proteins termed cancer/testis (CT) antigens, since expression outside of their naturally immune-privileged site can evoke an immune response. Despite extensive efforts to exploit CT antigens as immunotherapeutic targets, investigation of whether these proteins participate in tumorigenic processes has lagged. Here, we discuss emerging evidence that demonstrates that CT antigens can confer a selective advantage to tumor cells by promoting oncogenic processes or permitting evasion of tumor-suppressive mechanisms. These advances indicate the inherent flexibility of tumor cell regulatory networks to engage aberrantly expressed proteins to promote neoplastic behaviors, which could ultimately present novel therapeutic entry points.


Assuntos
Antígenos de Neoplasias/metabolismo , Carcinogênese/imunologia , Regulação Neoplásica da Expressão Gênica/imunologia , Neoplasias/imunologia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinogênese/genética , Modelos Animais de Doenças , Drosophila melanogaster , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Proteólise
15.
Cancer Res ; 78(21): 6196-6208, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30185546

RESUMO

Cancer testis antigens (CTA) are expressed in testis and placenta and anomalously activated in a variety of tumors. The mechanistic contribution of CTAs to neoplastic phenotypes remains largely unknown. Using a chemigenomics approach, we find that the CTA HORMAD1 correlates with resistance to the mitochondrial complex I inhibitor piericidin A in non-small cell lung cancer (NSCLC). Resistance was due to a reductive intracellular environment that attenuated the accumulation of free radicals. In human lung adenocarcinoma (LUAD) tumors, patients expressing high HORMAD1 exhibited elevated mutational burden and reduced survival. HORMAD1 tumors were enriched for genes essential for homologous recombination (HR), and HORMAD1 promoted RAD51-filament formation, but not DNA resection, during HR. Accordingly, HORMAD1 loss enhanced sensitivity to γ-irradiation and PARP inhibition, and HORMAD1 depletion significantly reduced tumor growth in vivo These results suggest that HORMAD1 expression specifies a novel subtype of LUAD, which has adapted to mitigate DNA damage. In this setting, HORMAD1 could represent a direct target for intervention to enhance sensitivity to DNA-damaging agents or as an immunotherapeutic target in patients.Significance: This study uses a chemigenomics approach to demonstrate that anomalous expression of the CTA HORMAD1 specifies resistance to oxidative stress and promotes HR to support tumor cell survival in NSCLC. Cancer Res; 78(21); 6196-208. ©2018 AACR.


Assuntos
Adenocarcinoma de Pulmão/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Proteínas de Ciclo Celular/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/diagnóstico , Células A549 , Adenocarcinoma de Pulmão/metabolismo , Animais , Antígenos de Neoplasias/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Dano ao DNA , Reparo do DNA , Feminino , Radicais Livres , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Mutagênicos , Transplante de Neoplasias , Estresse Oxidativo , Prognóstico , Recombinação Genética
16.
Autophagy ; 13(5): 969-970, 2017 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-28282258

RESUMO

Autophagy is a cellular degradation pathway that is essential to maintain cellular physiology, and deregulation of autophagy leads to multiple diseases in humans. In a recent study, we discovered that the protein kinase WNK1 (WNK lysine deficient protein kinase 1) is an inhibitor of autophagy. The loss of WNK1 increases both basal and starvation-induced autophagy. In addition, the depletion of WNK1 increases the activation of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex, which is required to induce autophagy. Moreover, the loss of WNK1 increases the expression of ULK1 (unc-51 like kinase 1), which is upstream of the PtdIns3K complex. It also increases the pro-autophagic phosphorylation of ULK1 at Ser555 and the activation of AMPK (AMP-activated protein kinase), which is responsible for that phosphorylation. The inhibition of AMPK by compound C decreases the magnitude of autophagy induction following WNK1 loss; however, it does not prevent autophagy induction. We found that the UVRAG (UV radiation resistance associated gene), which is a component of the PtdIns3K, binds to the N-terminal region of WNK1. Moreover, WNK1 partially colocalizes with UVRAG and this colocalization decreases when autophagy is stimulated in cells. The loss of WNK1 also alters the cellular distribution of UVRAG. The depletion of the downstream target of WNK1, OXSR1/OSR1 (oxidative-stress responsive 1) has no effect on autophagy, whereas the depletion of its relative STK39/SPAK (serine/threonine kinase 39) induces autophagy under nutrient-rich and starved conditions.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia/fisiologia , Transdução de Sinais/fisiologia , Proteína Quinase 1 Deficiente de Lisina WNK/metabolismo , Animais , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo
17.
Methods Enzymol ; 407: 259-68, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16757330

RESUMO

The Ras-regulatory network is a loosely defined composition of numerous Ras family members and effector pathways that couple to critical cell-regulatory processes. Investigators are increasingly turning to RNAi-mediated inhibition of gene expression as an effective tool to help generate authentic portraits of Ras protein function in general and to accurately characterize the contribution of Ras family members and Ras effectors to oncogenic transformation in particular. Here we provide detailed protocols for high-efficiency and high-throughput delivery of siRNAs to human cancer cell lines and primary human epithelial cells. In addition, we discuss appropriate controls and limitations for the use of RNAi to derive biologically relevant observations.


Assuntos
Transformação Celular Neoplásica , Interferência de RNA , Transdução de Sinais/fisiologia , Proteínas ras/fisiologia , Linhagem Celular Tumoral , Células HeLa , Humanos , Transfecção/métodos , Tripsina/metabolismo
18.
Mol Cell Biol ; 36(24): 3048-3057, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27697861

RESUMO

Triple-negative breast cancer (TNBC) is a highly heterogeneous disease with multiple, distinct molecular subtypes that exhibit unique transcriptional programs and clinical progression trajectories. Despite knowledge of the molecular heterogeneity of the disease, most patients are limited to generic, indiscriminate treatment options: cytotoxic chemotherapy, surgery, and radiation. To identify new intervention targets in TNBC, we used large-scale, loss-of-function screening to identify molecular vulnerabilities among different oncogenomic backgrounds. This strategy returned salt inducible kinase 2 (SIK2) as essential for TNBC survival. Genetic or pharmacological inhibition of SIK2 leads to increased autophagic flux in both normal-immortalized and tumor-derived cell lines. However, this activity causes cell death selectively in breast cancer cells and is biased toward the claudin-low subtype. Depletion of ATG5, which is essential for autophagic vesicle formation, rescued the loss of viability following SIK2 inhibition. Importantly, we find that SIK2 is essential for TNBC tumor growth in vivo Taken together, these findings indicate that claudin-low tumor cells rely on SIK2 to restrain maladaptive autophagic activation. Inhibition of SIK2 therefore presents itself as an intervention opportunity to reactivate this tumor suppressor mechanism.


Assuntos
Proteína 5 Relacionada à Autofagia/genética , Claudinas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Neoplasias de Mama Triplo Negativas/patologia , Animais , Autofagia , Proteína 5 Relacionada à Autofagia/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Transplante de Neoplasias , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo
19.
Oncotarget ; 7(12): 14708-26, 2016 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-26895102

RESUMO

Genes that are normally biased towards expression in the testis are often induced in tumor cells. These gametogenic genes, known as cancer-testis antigens (CTAs), have been extenstively investigated as targets for immunotherapy. However, despite their frequent detection, the degree to which CTAs support neoplastic invasion is poorly understood. Here, we find that the CTA genes SPANX-A/C/D and CTAG2 are coordinately induced in breast cancer cells and regulate distinct features of invasive behavior. Our functional analysis revealed that CTAG2 interacts with Pericentrin at the centrosome and is necessary for directional migration. Conversely, SPANX-A/C/D interacts with Lamin A/C at the inner nuclear membrane and is required for the formation of actin-rich cellular protrusions that reorganize the extracellular matrix. Importantly, SPANX-A/C/D was required for breast cancer cells to spontaneously metastasize to the lung, demonstrating that CTA reactivation can be critical for invasion dependent phenotypes in vivo. Moreover, elevated SPANX-A/C/D expression in breast cancer patient tumors correlated with poor outcome. Together, our results suggest that distinct CTAs promote tumor progression by regulating complementary cellular functions that are integrated together to induce invasive behavior.


Assuntos
Antígenos de Neoplasias/metabolismo , Antígenos de Superfície/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Animais , Apoptose , Neoplasias da Mama/metabolismo , Movimento Celular , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Estadiamento de Neoplasias , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Nat Commun ; 6: 8840, 2015 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-26567849

RESUMO

Tumours frequently activate genes whose expression is otherwise biased to the testis, collectively known as cancer-testis antigens (CTAs). The extent to which CTA expression represents epiphenomena or confers tumorigenic traits is unknown. In this study, to address this, we implemented a multidimensional functional genomics approach that incorporates 7 different phenotypic assays in 11 distinct disease settings. We identify 26 CTAs that are essential for tumor cell viability and/or are pathological drivers of HIF, WNT or TGFß signalling. In particular, we discover that Foetal and Adult Testis Expressed 1 (FATE1) is a key survival factor in multiple oncogenic backgrounds. FATE1 prevents the accumulation of the stress-sensing BH3-only protein, BCL-2-Interacting Killer (BIK), thereby permitting viability in the presence of toxic stimuli. Furthermore, ZNF165 promotes TGFß signalling by directly suppressing the expression of negative feedback regulatory pathways. This action is essential for the survival of triple negative breast cancer cells in vitro and in vivo. Thus, CTAs make significant direct contributions to tumour biology.


Assuntos
Antígenos de Neoplasias/genética , Carcinogênese/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/genética , Adenocarcinoma/genética , Adenocarcinoma/imunologia , Adenocarcinoma/mortalidade , Adenocarcinoma de Pulmão , Animais , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/mortalidade , Imunofluorescência , Células HCT116 , Células HEK293 , Humanos , Immunoblotting , Técnicas In Vitro , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/mortalidade , Proteínas de Membrana/genética , Camundongos Endogâmicos NOD , Proteínas Mitocondriais , Transplante de Neoplasias , Neoplasias/imunologia , Neoplasias/mortalidade , Prognóstico , Modelos de Riscos Proporcionais , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Proteína Smad7/genética , Fator de Crescimento Transformador beta/imunologia , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/mortalidade , Ubiquitina-Proteína Ligases/genética , Via de Sinalização Wnt
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