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1.
Sci Adv ; 8(42): eabq8297, 2022 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-36269833

RESUMO

Fumarate hydratase (FH) is a mitochondrial enzyme that catalyzes the reversible hydration of fumarate to malate in the tricarboxylic acid (TCA) cycle. Germline mutations of FH lead to hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a cancer syndrome characterized by a highly aggressive form of renal cancer. Although HLRCC tumors metastasize rapidly, FH-deficient mice develop premalignant cysts in the kidneys, rather than carcinomas. How Fh1-deficient cells overcome these tumor-suppressive events during transformation is unknown. Here, we perform a genome-wide CRISPR-Cas9 screen to identify genes that, when ablated, enhance the proliferation of Fh1-deficient cells. We found that the depletion of the histone cell cycle regulator (HIRA) enhances proliferation and invasion of Fh1-deficient cells in vitro and in vivo. Mechanistically, Hira loss activates MYC and its target genes, increasing nucleotide metabolism specifically in Fh1-deficient cells, independent of its histone chaperone activity. These results are instrumental for understanding mechanisms of tumorigenesis in HLRCC and the development of targeted treatments for patients.

2.
Mod Pathol ; 34(11): 2009-2019, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34155350

RESUMO

Dedifferentiation and transdifferentiation are rare and only poorly understood phenomena in cutaneous melanoma. To study this disease more comprehensively we have retrieved 11 primary cutaneous melanomas from our pathology archives showing biphasic features characterized by a conventional melanoma and additional areas of de-/trans-differentiation as defined by a lack of immunohistochemical expression of all conventional melanocytic markers (S-100 protein, SOX10, Melan-A, and HMB-45). The clinical, histologic, and immunohistochemical findings were recorded and follow-up was obtained. The patients were mostly elderly (median: 81 years; range: 42-86 years) without significant gender predilection, and the sun-exposed skin of the head and neck area was most commonly affected. The tumors were deeply invasive with a mean depth of 7 mm (range: 4-80 mm). The dedifferentiated component showed atypical fibroxanthoma-like features in the majority of cases (7), while additional rhabdomyosarcomatous and epithelial transdifferentiation was noted histologically and/or immunohistochemically in two tumors each. The background conventional melanoma component was of desmoplastic (4), superficial spreading (3), nodular (2), lentigo maligna (1), or spindle cell (1) types. For the seven patients with available follow-up data (median follow-up period of 25 months; range: 8-36 months), two died from their disease, and three developed metastases. Next-generation sequencing of the cohort revealed somatic mutations of established melanoma drivers including mainly NF1 mutations (5) in the conventional component, which was also detected in the corresponding de-/trans-differentiated component. In summary, the diagnosis of primary cutaneous de-/trans-differentiated melanoma is challenging and depends on the morphologic identification of conventional melanoma. Molecular analysis is diagnostically helpful as the mutated gene profile is shared between the conventional and de-/trans-differentiated components. Importantly, de-/trans-differentiation does not appear to confer a more aggressive behavior.


Assuntos
Genômica , Melanoma/patologia , Neurofibromina 1/genética , Neoplasias Cutâneas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Diferenciação Celular , DNA de Neoplasias/genética , Diagnóstico Diferencial , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Melanoma/genética , Melanoma/metabolismo , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo
3.
Commun Biol ; 4(1): 395, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758365

RESUMO

Melanoma represents ~5% of all cutaneous malignancies, yet accounts for the majority of skin cancer deaths due to its propensity to metastasise. To develop new therapies, novel target molecules must to be identified and the accessibility of cell surface proteins makes them attractive targets. Using CRISPR activation technology, we screened a library of guide RNAs targeting membrane protein-encoding genes to identify cell surface molecules whose upregulation enhances the metastatic pulmonary colonisation capabilities of tumour cells in vivo. We show that upregulated expression of the cell surface protein LRRN4CL led to increased pulmonary metastases in mice. Critically, LRRN4CL expression was elevated in melanoma patient samples, with high expression levels correlating with decreased survival. Collectively, our findings uncover an unappreciated role for LRRN4CL in the outcome of melanoma patients and identifies a potential therapeutic target and biomarker.


Assuntos
Biomarcadores Tumorais/metabolismo , Sistemas CRISPR-Cas , Neoplasias Pulmonares/metabolismo , Melanoma Experimental/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Movimento Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Masculino , Melanoma Experimental/genética , Melanoma Experimental/secundário , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Invasividade Neoplásica , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Regulação para Cima
4.
Nat Commun ; 12(1): 1302, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33637726

RESUMO

Genetic redundancy has evolved as a way for human cells to survive the loss of genes that are single copy and essential in other organisms, but also allows tumours to survive despite having highly rearranged genomes. In this study we CRISPR screen 1191 gene pairs, including paralogues and known and predicted synthetic lethal interactions to identify 105 gene combinations whose co-disruption results in a loss of cellular fitness. 27 pairs influence fitness across multiple cell lines including the paralogues FAM50A/FAM50B, two genes of unknown function. Silencing of FAM50B occurs across a range of tumour types and in this context disruption of FAM50A reduces cellular fitness whilst promoting micronucleus formation and extensive perturbation of transcriptional programmes. Our studies reveal the fitness effects of FAM50A/FAM50B in cancer cells.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Genoma , Proteínas/genética , Animais , Apoptose , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Técnicas de Inativação de Genes , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Ligação a RNA/genética , Transcriptoma
5.
Life Sci Alliance ; 3(12)2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33033111

RESUMO

Docetaxel chemotherapy in metastatic prostate cancer offers only a modest survival benefit because of emerging resistance. To identify candidate therapeutic gene targets, we applied a murine prostate cancer orthograft model that recapitulates clinical invasive prostate cancer in a genome-wide CRISPR/Cas9 screen under docetaxel treatment pressure. We identified 17 candidate genes whose suppression may enhance the efficacy of docetaxel, with transcription elongation factor A-like 1 (Tceal1) as the top candidate. TCEAL1 function is not fully characterised; it may modulate transcription in a promoter dependent fashion. Suppressed TCEAL1 expression in multiple human prostate cancer cell lines enhanced therapeutic response to docetaxel. Based on gene set enrichment analysis from transcriptomic data and flow cytometry, we confirmed that loss of TCEAL1 in combination with docetaxel leads to an altered cell cycle profile compared with docetaxel alone, with increased subG1 cell death and increased polyploidy. Here, we report the first in vivo genome-wide treatment sensitisation CRISPR screen in prostate cancer, and present proof of concept data on TCEAL1 as a candidate for a combinational strategy with the use of docetaxel.


Assuntos
Proteínas de Ligação a DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias da Próstata/genética , Fatores de Transcrição/genética , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Proteínas de Ligação a DNA/metabolismo , Docetaxel/farmacologia , Regulação Neoplásica da Expressão Gênica/genética , Engenharia Genética/métodos , Masculino , Camundongos , Camundongos Nus , Neoplasias da Próstata/metabolismo , Taxoides/farmacologia , Fatores de Transcrição/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
6.
Cancer Res ; 80(3): 576-590, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31719098

RESUMO

Inhibition of the androgen receptor (AR) is the main strategy to treat advanced prostate cancers. AR-independent treatment-resistant prostate cancer is a major unresolved clinical problem. Patients with prostate cancer with alterations in canonical WNT pathway genes, which lead to ß-catenin activation, are refractory to AR-targeted therapies. Here, using clinically relevant murine prostate cancer models, we investigated the significance of ß-catenin activation in prostate cancer progression and treatment resistance. ß-Catenin activation, independent of the cell of origin, cooperated with Pten loss to drive AR-independent castration-resistant prostate cancer. Prostate tumors with ß-catenin activation relied on the noncanonical WNT ligand WNT5a for sustained growth. WNT5a repressed AR expression and maintained the expression of c-Myc, an oncogenic effector of ß-catenin activation, by mediating nuclear localization of NFκBp65 and ß-catenin. Overall, WNT/ß-catenin and AR signaling are reciprocally inhibited. Therefore, inhibiting WNT/ß-catenin signaling by limiting WNT secretion in concert with AR inhibition may be useful for treating prostate cancers with alterations in WNT pathway genes. SIGNIFICANCE: Targeting of both AR and WNT/ß-catenin signaling may be required to treat prostate cancers that exhibit alterations of the WNT pathway.


Assuntos
Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , PTEN Fosfo-Hidrolase/deficiência , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/metabolismo , Proteína Wnt-5a/metabolismo , beta Catenina/metabolismo , Antagonistas de Receptores de Andrógenos/farmacologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Humanos , Masculino , Camundongos , Prognóstico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptores Androgênicos/genética , Taxa de Sobrevida , Células Tumorais Cultivadas , Proteína Wnt-5a/genética , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genética
7.
EMBO Mol Med ; 10(4)2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29540470

RESUMO

Metastatic castration-resistant prostate cancer (mCRPC) is a lethal form of treatment-resistant prostate cancer and poses significant therapeutic challenges. Deregulated receptor tyrosine kinase (RTK) signalling mediated by loss of tumour suppressor Sprouty2 (SPRY2) is associated with treatment resistance. Using pre-clinical human and murine mCRPC models, we show that SPRY2 deficiency leads to an androgen self-sufficient form of CRPC Mechanistically, HER2-IL6 signalling axis enhances the expression of androgen biosynthetic enzyme HSD3B1 and increases SRB1-mediated cholesterol uptake in SPRY2-deficient tumours. Systemically, IL6 elevated the levels of circulating cholesterol by inducing host adipose lipolysis and hepatic cholesterol biosynthesis. SPRY2-deficient CRPC is dependent on cholesterol bioavailability and SRB1-mediated tumoral cholesterol uptake for androgen biosynthesis. Importantly, treatment with ITX5061, a clinically safe SRB1 antagonist, decreased treatment resistance. Our results indicate that cholesterol transport blockade may be effective against SPRY2-deficient CRPC.


Assuntos
Interleucina-6/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptores Depuradores/metabolismo , Animais , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas de Membrana/genética , Camundongos Nus , Fenilenodiaminas/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Proteínas Serina-Treonina Quinases , Reação em Cadeia da Polimerase em Tempo Real , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptores Depuradores Classe B/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Sulfonamidas/uso terapêutico
8.
Oncotarget ; 6(35): 37724-36, 2015 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-26462181

RESUMO

UNLABELLED: The androgen receptor (AR) is a key transcription factor in the initiation and progression of prostate cancer (PC) and is a major therapeutic target for the treatment of advanced disease. Unfortunately, current therapies are not curative for castration resistant PC and a better understanding of AR regulation could identify novel therapeutic targets and biomarkers to aid treatment of this disease. The AR is known to be regulated by a number of post-translational modifications and we have recently identified the deubiquitinating enzyme Usp12 as a positive regulator of AR. We determined that Usp12 deubiquitinates the AR resulting in elevated receptor stability and activity. Furthermore, Usp12 silencing was shown to reduce proliferation of PC cells.Usp12 is known to require the co-factors Uaf-1 and WDR20 for catalytic activity. In this report we focus further on the role of Uaf-1 and WDR20 in Usp12 regulation and investigate if these co-factors are also required for controlling AR activity. Firstly, we confirm the presence of the Usp12/Uaf-1/WDR20 complex in PC cells and demonstrate the importance of Uaf-1 and WDR20 for Usp12 stabilisation. Consequently, we show that individual silencing of either Uaf-1 or WDR20 is sufficient to abrogate the activity of the Usp12 complex and down-regulate AR-mediated transcription via receptor destabilisation resulting in increased apoptosis and decreased colony forming ability of PC cells. Moreover, expression of both Uaf-1 and WDR20 is higher in PC tissue compared to benign controls. Overall these results highlight the potential importance of the Usp12/Uaf-1/WDR20 complex in AR regulation and PC progression. HIGHLIGHTS: • Androgen receptor is a key transcriptional regulator in prostate cancer • Usp12/Uaf-1/WDR20 complex plays a crucial role in androgen receptor stability and activity • Destabilising an individual Usp12/Uaf-1/WDR20 complex member reduces the protein levels of the whole complex and diminishes androgen receptor activity • Protein levels of all members of the Usp12/Uaf-1/WDR20 complex are significantly increased in PC.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/genética , Apoptose , Western Blotting , Proteínas de Transporte/genética , Proliferação de Células , Imunoprecipitação da Cromatina , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoprecipitação , Masculino , Proteínas Nucleares/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Transcrição Gênica , Células Tumorais Cultivadas , Proteases Específicas de Ubiquitina/genética
9.
Oncotarget ; 5(16): 7081-92, 2014 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-25216524

RESUMO

The androgen receptor (AR) is a transcription factor involved in prostate cell growth, homeostasis and transformation regulated by post-translational modifications, including ubiquitination. We have recently reported that AR is deubiquitinated and stabilised by Usp12 resulting in increased transcriptional activity. In this study we have investigated the relationship between Usp12, PHLPP and PHLPPL tumour suppressors in the regulation of AR transcriptional activity in prostate cancer (PC). PHLPP and PHLPPL are pro-apoptotic phosphatases that dephosphorylate and subsequently deactivate Akt. Phosphorylated Akt is reported to deactivate AR in PC by phosphorylation at Ser213 and Ser791 leading to ligand dissociation and AR degradation. In contrast, PHLPP- and PHLPPL-mediated dephosphorylation and inactivation of Akt elevates the levels of active AR. In this report we demonstrate that Usp12, in complex with Uaf-1 and WDR20, directly deubiquitinates and stabilises the Akt phosphatases PHLPP and PHLPPL resulting in decreased levels of active pAkt. Decreased pAkt in turn down-regulates AR Ser213 phosphorylation resulting in enhanced receptor stability and transcriptional activity. Additionally, we observe that depleting Usp12 sensitises PC cells to therapies aimed at Akt inhibition irrespectively of their sensitivity to androgen ablation therapy. We propose that Usp12 inhibition could offer a therapeutic alternative for castration resistant prostate cancer.


Assuntos
Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Androgênicos/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Chlorocebus aethiops , Células HEK293 , Humanos , Masculino , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Receptores Androgênicos/genética , Transdução de Sinais , Ubiquitina Tiolesterase/genética , Ubiquitinação
10.
J Biol Chem ; 288(45): 32641-32650, 2013 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-24056413

RESUMO

The androgen receptor (AR), a member of the nuclear receptor family, is a transcription factor involved in prostate cell growth, homeostasis, and transformation. AR is a key protein in growth and development of both normal and malignant prostate, making it a common therapeutic target in prostate cancer. AR is regulated by an interplay of multiple post-translational modifications including ubiquitination. We and others have shown that the AR is ubiquitinated by a number of E3 ubiquitin ligases, including MDM2, CHIP, and NEDD4, which can result in its proteosomal degradation or enhanced transcriptional activity. As ubiquitination of AR causes a change in AR activity or stability and impacts both survival and growth of prostate cancer cells, deubiquitination of these sites has an equally important role. Hence, deubiquitinating enzymes could offer novel therapeutic targets. We performed an siRNA screen to identify deubiquitinating enzymes that regulate AR; in that screen ubiquitin-specific protease 12 (Usp12) was identified as a novel positive regulator of AR. Usp12 is a poorly characterized protein with few known functions and requires the interaction with two cofactors, Uaf-1 and WDR20, for its enzymatic activity. In this report we demonstrate that Usp12, in complex with Uaf-1 and WDR20, deubiquitinates the AR to enhance receptor stability and transcriptional activity. Our data show that Usp12 acts in a pro-proliferative manner by stabilizing AR and enhancing its cellular function.


Assuntos
Proliferação de Células , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Animais , Células COS , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Masculino , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/genética , Estabilidade Proteica , Receptores Androgênicos/genética , Ubiquitina Tiolesterase/genética , Ubiquitina-Proteína Ligases/genética
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