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1.
J Biol Chem ; 299(1): 102774, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36481270

RESUMEN

Hexanucleotide expansion mutations in C9ORF72 are a frequent cause of amyotrophic lateral sclerosis. We previously reported that long arginine-rich dipeptide repeats (DPRs), mimicking abnormal proteins expressed from the hexanucleotide expansion, caused translation stalling when expressed in cell culture models. Whether this stalling provides a mechanism of pathogenicity remains to be determined. Here, we explored the molecular features of DPR-induced stalling and examined whether known mechanisms such as ribosome quality control (RQC) regulate translation elongation on sequences that encode arginine-rich DPRs. We demonstrate that arginine-rich DPRs lead to stalling in a length-dependent manner, with lengths longer than 40 repeats invoking severe translation arrest. Mutational screening of 40×Gly-Xxx DPRs shows that stalling is most pronounced when Xxx is a charged amino acid (Arg, Lys, Glu, or Asp). Through a genome-wide knockout screen, we find that genes regulating stalling on polyadenosine mRNA coding for poly-Lys, a canonical RQC substrate, act differently in the case of arginine-rich DPRs. Indeed, these findings point to a limited scope for natural regulatory responses to resolve the arginine-rich DPR stalls, even though the stalls may be sensed, as evidenced by an upregulation of RQC gene expression. These findings therefore implicate arginine-rich DPR-mediated stalled ribosomes as a source of stress and toxicity and may be a crucial component in pathomechanisms.


Asunto(s)
Esclerosis Amiotrófica Lateral , Humanos , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/fisiopatología , Arginina/metabolismo , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Dipéptidos/química , Ribosomas/genética , Ribosomas/metabolismo , Técnicas de Inactivación de Genes , Mutación , Regulación hacia Arriba
2.
Nat Immunol ; 13(6): 543-550, 2012 Apr 29.
Artículo en Inglés | MEDLINE | ID: mdl-22544393

RESUMEN

Type I interferon is an integral component of the antiviral response, and its production is tightly controlled at the levels of transcription and translation. The eukaryotic translation-initiation factor eIF4E is a rate-limiting factor whose activity is regulated by phosphorylation of Ser209. Here we found that mice and fibroblasts in which eIF4E cannot be phosphorylated were less susceptible to virus infection. More production of type I interferon, resulting from less translation of Nfkbia mRNA (which encodes the inhibitor IκBα), largely explained this phenotype. The lower abundance of IκBα resulted in enhanced activity of the transcription factor NF-κB, which promoted the production of interferon-ß (IFN-ß). Thus, regulated phosphorylation of eIF4E has a key role in antiviral host defense by selectively controlling the translation of an mRNA that encodes a critical suppressor of the innate antiviral response.


Asunto(s)
Factor 4E Eucariótico de Iniciación/metabolismo , Interferón Tipo I/biosíntesis , FN-kappa B/metabolismo , Estomatitis Vesicular/inmunología , Virus de la Estomatitis Vesicular Indiana/fisiología , Animales , Ensayo de Cambio de Movilidad Electroforética , Factor 4E Eucariótico de Iniciación/inmunología , Femenino , Proteínas I-kappa B/biosíntesis , Proteínas I-kappa B/genética , Proteínas I-kappa B/inmunología , Inmunidad Innata/inmunología , Immunoblotting , Interferón Tipo I/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Inhibidor NF-kappaB alfa , FN-kappa B/inmunología , Fosforilación , Biosíntesis de Proteínas , ARN Mensajero/química , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Organismos Libres de Patógenos Específicos , Estomatitis Vesicular/genética , Estomatitis Vesicular/metabolismo , Estomatitis Vesicular/virología , Virus de la Estomatitis Vesicular Indiana/inmunología , Replicación Viral
3.
EMBO J ; 38(23): e101323, 2019 12 02.
Artículo en Inglés | MEDLINE | ID: mdl-31556460

RESUMEN

Estrogen receptor alpha (ERα) activity is associated with increased cancer cell proliferation. Studies aiming to understand the impact of ERα on cancer-associated phenotypes have largely been limited to its transcriptional activity. Herein, we demonstrate that ERα coordinates its transcriptional output with selective modulation of mRNA translation. Importantly, translational perturbations caused by depletion of ERα largely manifest as "translational offsetting" of the transcriptome, whereby amounts of translated mRNAs and corresponding protein levels are maintained constant despite changes in mRNA abundance. Transcripts whose levels, but not polysome association, are reduced following ERα depletion lack features which limit translation efficiency including structured 5'UTRs and miRNA target sites. In contrast, mRNAs induced upon ERα depletion whose polysome association remains unaltered are enriched in codons requiring U34-modified tRNAs for efficient decoding. Consistently, ERα regulates levels of U34-modifying enzymes and thereby controls levels of U34-modified tRNAs. These findings unravel a hitherto unprecedented mechanism of ERα-dependent orchestration of transcriptional and translational programs that may be a pervasive mechanism of proteome maintenance in hormone-dependent cancers.


Asunto(s)
Neoplasias de la Mama/genética , Receptor alfa de Estrógeno/genética , Regulación Neoplásica de la Expresión Génica , Polirribosomas/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Células MCF-7 , Polirribosomas/metabolismo , ARN Mensajero/metabolismo , Transducción de Señal , Activación Transcripcional
4.
Nucleic Acids Res ; 47(12): e70, 2019 07 09.
Artículo en Inglés | MEDLINE | ID: mdl-30926999

RESUMEN

mRNA translation plays an evolutionarily conserved role in homeostasis and when dysregulated contributes to various disorders including metabolic and neurological diseases and cancer. Notwithstanding that optimal and universally applicable methods are critical for understanding the complex role of translational control under physiological and pathological conditions, approaches to analyze translatomes are largely underdeveloped. To address this, we developed the anota2seq algorithm which outperforms current methods for statistical identification of changes in translation. Notably, in contrast to available analytical methods, anota2seq also allows specific identification of an underappreciated mode of gene expression regulation whereby translation acts as a buffering mechanism which maintains protein levels despite fluctuations in corresponding mRNA abundance ('translational buffering'). Thus, the universal anota2seq algorithm allows efficient and hitherto unprecedented interrogation of translatomes which is anticipated to advance knowledge regarding the role of translation in homeostasis and disease.


Asunto(s)
Algoritmos , Biosíntesis de Proteínas , Interpretación Estadística de Datos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas/análisis , Proteínas Ribosómicas , Ribosomas , Análisis de Secuencia de ARN , Transcriptoma
5.
J Biol Chem ; 293(23): 8874-8885, 2018 06 08.
Artículo en Inglés | MEDLINE | ID: mdl-29695504

RESUMEN

Inhibition of the metabolic regulator AMP-activated protein kinase (AMPK) is increasingly being investigated for its therapeutic potential in diseases where AMPK hyperactivity results in poor prognoses, as in established cancers and neurodegeneration. However, AMPK-inhibitory tool compounds are largely limited to compound C, which has a poor selectivity profile. Here we identify the pyrimidine derivative SBI-0206965 as a direct AMPK inhibitor. SBI-0206965 inhibits AMPK with 40-fold greater potency and markedly lower kinase promiscuity than compound C and inhibits cellular AMPK signaling. Biochemical characterization reveals that SBI-0206965 is a mixed-type inhibitor. A co-crystal structure of the AMPK kinase domain/SBI-0206965 complex shows that the drug occupies a pocket that partially overlaps the ATP active site in a type IIb inhibitor manner. SBI-0206965 has utility as a tool compound for investigating physiological roles for AMPK and provides fresh impetus to small-molecule AMPK inhibitor therapeutic development.


Asunto(s)
Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Benzamidas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Benzamidas/química , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas/química , Pirimidinas/química
6.
Genome Res ; 26(5): 636-48, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-26984228

RESUMEN

The diversity of MTOR-regulated mRNA translation remains unresolved. Whereas ribosome-profiling suggested that MTOR almost exclusively stimulates translation of the TOP (terminal oligopyrimidine motif) and TOP-like mRNAs, polysome-profiling indicated that MTOR also modulates translation of mRNAs without the 5' TOP motif (non-TOP mRNAs). We demonstrate that in ribosome-profiling studies, detection of MTOR-dependent changes in non-TOP mRNA translation was obscured by low sensitivity and methodology biases. Transcription start site profiling using nano-cap analysis of gene expression (nanoCAGE) revealed that not only do many MTOR-sensitive mRNAs lack the 5' TOP motif but that 5' UTR features distinguish two functionally and translationally distinct subsets of MTOR-sensitive mRNAs: (1) mRNAs with short 5' UTRs enriched for mitochondrial functions, which require EIF4E but are less EIF4A1-sensitive; and (2) long 5' UTR mRNAs encoding proliferation- and survival-promoting proteins, which are both EIF4E- and EIF4A1-sensitive. Selective inhibition of translation of mRNAs harboring long 5' UTRs via EIF4A1 suppression leads to sustained expression of proteins involved in respiration but concomitant loss of those protecting mitochondrial structural integrity, resulting in apoptosis. Conversely, simultaneous suppression of translation of both long and short 5' UTR mRNAs by MTOR inhibitors results in metabolic dormancy and a predominantly cytostatic effect. Thus, 5' UTR features define different modes of MTOR-sensitive translation of functionally distinct subsets of mRNAs, which may explain the diverse impact of MTOR and EIF4A inhibitors on neoplastic cells.


Asunto(s)
Regiones no Traducidas 5'/fisiología , Factor 4E Eucariótico de Iniciación/metabolismo , Biosíntesis de Proteínas/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Apoptosis/fisiología , Femenino , Humanos , Células MCF-7
7.
Mol Hum Reprod ; 24(3): 111-122, 2018 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-29361143

RESUMEN

STUDY QUESTION: What is the role of epididymal cysteine-rich secretory proteins (CRISPs) in male fertility? SUMMARY ANSWER: While epididymal CRISPs are not absolutely required for male fertility, they are required for optimal sperm function. WHAT IS KNOWN ALREADY: CRISPs are members of the CRISP, Antigen 5 and Pathogenesis related protein 1 (CAP) superfamily and are characterized by the presence of an N-terminal CAP domain and a C-terminal CRISP domain. CRISPs are highly enriched in the male reproductive tract of mammals, including in the epididymis. Within humans there is one epididymal CRISP, CRISP1, whereas in mice there are two, CRISP1 and CRISP4. STUDY DESIGN, SIZE, DURATION: In order to define the role of CRISPs within the epididymis, Crisp1 and Crisp4 knockout mouse lines were produced then interbred to produce Crisp1 and 4 double knockout (DKO) mice, wherein the expression of all epididymal CRISPs was ablated. Individual and DKO models were then assessed, relative to their own strain-specific wild type littermates for fertility, and sperm output and functional competence at young (10-12 weeks of age) and older ages (22-24 weeks). Crisp1 and 4 DKO and control mice were also compared for their ability to bind to the zona pellucida and achieve fertilization. PARTICIPANTS/MATERIALS, SETTING, METHODS: Knockout mouse production was achieved using modified embryonic stem cells and standard methods. The knockout of individual genes was confirmed at a mRNA (quantitative PCR) and protein (immunochemistry) level. Fertility was assessed using breeding experiments and a histological assessment of testes and epididymal tissue. Sperm functional competence was assessed using a computer assisted sperm analyser, induction of the acrosome reaction using progesterone followed by staining for acrosome contents, using immunochemical and western blotting to assess the ability of sperm to manifest tyrosine phosphorylation under capacitating conditions and using sperm-zona pellucida binding assays and IVF methods. A minimum of three biological replicates were used per assay and per genotype. MAIN RESULTS AND THE ROLE OF CHANCE: While epididymal CRISPs are not absolutely required for male fertility, their production results in enhanced sperm function and, depending on context, CRISP1 and CRISP4 act redundantly or autonomously. Specifically, CRISP1 is the most important CRISP in the establishment of normally motile sperm, whereas CRISP4 acts to enhance capacitation-associated tyrosine phosphorylation, and CRISP1 and CRISP4 act together to establish normal acrosome function. Both are required to achieve optimal sperm-egg interaction. The presence of immune infiltrates into the epididymis of older, but not younger, DKO animals also suggests epididymal CRISPs function to produce an immune privileged environment for maturing sperm within the epididymis. LIMITATIONS REASONS FOR CAUTION: Caution should be displayed in the translation of mouse-derived data into the human wherein the histology of the epididymis is someone what different. The mice used in the study were housed in a specific pathogen-free environment and were thus not exposed to the full range of environmental challenges experienced by wild mice or humans. As such, the role of CRISPs in the maintenance of an immune privileged environment, for example, may be understated. WIDER IMPLICATIONS OF THE FINDINGS: The combined deletion of Crisp1 and Crisp4 in mice is equivalent to the removal of all CRISP expression in humans. As such, these data suggest that mammalian CRISPs, including that in humans, function to enhance sperm function and thus male fertility. These data also suggest that in the presence of an environmental challenge, CRISPs help to maintain an immune privileged environment and thus, protect against immune-mediated male infertility. LARGE SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTEREST(S): This study was funded by the National Health and Medical Research Council, the Victorian Cancer Agency and a scholarship from the Chinese Scholarship Council. The authors have no conflicts of interest to declare.


Asunto(s)
Epidídimo/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Plasma Seminal/metabolismo , Maduración del Esperma/fisiología , Acrosoma/metabolismo , Acrosoma/fisiología , Animales , Línea Celular , Femenino , Humanos , Masculino , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Proteínas de Plasma Seminal/genética , Maduración del Esperma/genética
8.
RNA Biol ; 14(10): 1299-1305, 2017 10 03.
Artículo en Inglés | MEDLINE | ID: mdl-28277937

RESUMEN

Translation is fundamental for many biologic processes as it enables cells to rapidly respond to stimuli without requiring de novo mRNA synthesis. The mammalian/mechanistic target of rapamycin (mTOR) is a key regulator of translation. Although mTOR affects global protein synthesis, translation of a subset of mRNAs appears to be exceptionally sensitive to changes in mTOR activity. Recent efforts to catalog these mTOR-sensitive mRNAs resulted in conflicting results. Whereas ribosome-profiling almost exclusively identified 5'-terminal oligopyrimidine (TOP) mRNAs as mTOR-sensitive, polysome-profiling suggested that mTOR also regulates translation of non-TOP mRNAs. This inconsistency was explained by analytical and technical biases limiting the efficiency of ribosome-profiling in detecting mRNAs showing differential translation. Moreover, genome-wide characterization of 5'UTRs of non-TOP mTOR-sensitive mRNAs revealed 2 subsets of transcripts which differ in their requirement for translation initiation factors and biologic functions. We summarize these recent advances and their impact on the understanding of mTOR-sensitive translation.


Asunto(s)
Biosíntesis de Proteínas , Ribosomas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Regiones no Traducidas 5' , Animales , Regulación de la Expresión Génica , Humanos , ARN Mensajero/química , ARN Mensajero/genética
9.
J Pathol ; 234(1): 86-98, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25042571

RESUMEN

Prostate cancer is hormone-dependent and regulated by androgens as well as oestrogens. The tumour microenvironment also provides regulatory control, but the balance and interplay between androgens and oestrogens at the human prostate tumour interface is unknown. This study reveals a central and dominant role for oestrogen in the microenvironment, fuelling a pro-tumourigenic loop of inflammatory cytokines involving recruitment of mast cells by carcinoma-associated fibroblasts (CAFs). Mast cell numbers were increased in human PCa clinical specimens, specifically within the peritumoural stroma. Human mast cells were also shown to express ERα and ERß, with oestradiol directly stimulating mast cell proliferation and migration as well as altered cytokine/chemokine expression. There was a significant shift in the oestrogen:androgen balance in CAFs versus normal prostatic fibroblasts (NPFs), with a profound increase to ER:AR expression. Androgen signalling is also reduced in CAFs, while ERα and ERß transcriptional activity is not, allowing oestrogen to dictate hormone action in the tumour microenvironment. Gene microarray analyses identified CXCL12 as a major oestrogen-driven target gene in CAFs, and CAFs recruit mast cells via CXCL12 in a CXCR4-dependent manner. Collectively, these data reveal multicellular oestrogen action in the tumour microenvironment and show dominant oestrogen, rather than androgen, signalling at the prostatic tumour interface.


Asunto(s)
Carcinoma/patología , Quimiocina CXCL12/genética , Estrógenos/metabolismo , Neoplasias de la Próstata/patología , Receptores CXCR4/genética , Carcinoma/metabolismo , Comunicación Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Quimiocina CXCL12/metabolismo , Quimiocinas/genética , Quimiocinas/metabolismo , Citocinas/genética , Citocinas/metabolismo , Retroalimentación Fisiológica , Fibroblastos/metabolismo , Fibroblastos/patología , Perfilación de la Expresión Génica , Humanos , Masculino , Mastocitos/metabolismo , Mastocitos/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Receptores CXCR4/metabolismo , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Microambiente Tumoral
10.
J Nucl Med ; 65(8): 1231-1238, 2024 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-38991752

RESUMEN

[177Lu]Lu-PSMA is an effective class of therapy for patients with metastatic castration-resistant prostate cancer (mCRPC); however, progression is inevitable. The limited durability of response may be partially explained by the presence of micrometastatic deposits, which are energy-sheltered and receive low absorbed radiation with 177Lu due to the approximately 0.7-mm mean pathlength. 161Tb has abundant emission of Auger and conversion electrons that deposit a higher concentration of radiation over a shorter path, particularly to single tumor cells and micrometastases. 161Tb has shown in vitro and in vivo efficacy superior to that of 177Lu. We aim to demonstrate that [161Tb]Tb-PSMA-I&T will deliver effective radiation to sites of metastatic prostate cancer with an acceptable safety profile. Methods: This single-center, single-arm, phase I/II trial will recruit 30 patients with mCRPC. Key eligibility criteria include a diagnosis of mCRPC with progression after at least one line of taxane chemotherapy (unless medically unsuitable) and androgen receptor pathway inhibitor; prostate-specific membrane antigen-positive disease on [68Ga]Ga-PSMA-11 or [18F]DCFPyL PET/CT (SUVmax ≥ 20); no sites of discordance on [18F]FDG PET/CT; adequate bone marrow, hepatic, and renal function; an Eastern Cooperative Oncology Group performance status of no more than 2, and no prior treatment with another radioisotope. The dose escalation is a 3 + 3 design to establish the safety of 3 prespecified activities of [161Tb]Tb-PSMA-I&T (4.4, 5.5, and 7.4 GBq). The maximum tolerated dose will be defined as the highest activity level at which a dose-limiting toxicity occurs in fewer than 2 of 6 participants. The dose expansion will include 24 participants at the maximum tolerated dose. Up to 6 cycles of [161Tb]Tb-PSMA-I&T will be administered intravenously every 6 wk, with each subsequent activity reduced by 0.4 GBq. The coprimary objectives are to establish the maximum tolerated dose and safety profile (Common Terminology Criteria for Adverse Events version 5.0) of [161Tb]Tb-PSMA-I&T. Secondary objectives include measuring absorbed radiation dose (Gy), evaluating antitumor activity (prostate-specific antigen 50% response rate, radiographic and prostate-specific antigen progression-free survival, overall survival, objective response rate), and evaluating pain (Brief Pain Inventory-Short Form) and health-related quality of life (Functional Assessment of Cancer Therapy-Prostate and Functional Assessment of Cancer Therapy-Radionuclide Therapy). Conclusion: Enrollment was completed in February 2024. Patients are still receiving [161Tb]Tb-PSMA-I&T.


Asunto(s)
Metástasis de la Neoplasia , Neoplasias de la Próstata Resistentes a la Castración , Anciano , Humanos , Masculino , Persona de Mediana Edad , Ligandos , Neoplasias de la Próstata Resistentes a la Castración/radioterapia , Neoplasias de la Próstata Resistentes a la Castración/patología , Radiofármacos/uso terapéutico , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto
11.
Prostate ; 73(16): 1810-23, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24105601

RESUMEN

BACKGROUND: Contribution of stromal Hedgehog (Hh) signaling is evident in the prostate gland in mice, but needs translation to human tissues if Hh therapeutics are to be used effectively. Our goal was to determine if primary human prostate fibroblasts contain cilia, and respond to prostate Hh signaling. METHODS: Primary human prostate cancer-associated (CAFs), and adjacent non-malignant (NPFs) fibroblasts isolated from human tissue specimens were analyzed using immunofluorescence, real-time PCR, and available array data. Cell culture and tissue recombination were used to determine responsiveness of human fibroblasts to Hh pathway manipulation and the paracrine effects of stromal Hh signaling, respectively. RESULTS: Prostatic fibroblasts were capable of forming primary cilia, with the capacity for active Hh signaling as seen by Smo co-localization to the tip of the primary cilium. Expression of genes known to represent a signature of active Hh signaling in the prostate (especially Fgf5 and Igfbp6) were increased in CAFs compared to NPFs. The level of canonical Hh genes and prostate Hh signature genes were rarely synchronous; with lower doses of Purmorphamine/BMS-833923 regulating canonical transcription factors, and higher doses effecting prostate Hh signature genes. Grafts consisting of NPFs with constitutively active Hh signaling induced increased proliferation and dedifferentiation of adjacent non-malignant BPH-1 epithelial cells. CONCLUSIONS: These data show that human prostatic fibroblasts have the capacity for Hh signaling and manipulation. Increased expression of a signature of prostatic Hh genes in the prostate tumor microenvironment suggests a role in the epithelial transformations driving prostate cancer (PCa).


Asunto(s)
Comunicación Celular/fisiología , Diferenciación Celular/fisiología , Proliferación Celular , Proteínas Hedgehog/fisiología , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/fisiopatología , Transducción de Señal/fisiología , Animales , Benzamidas/farmacología , Transformación Celular Neoplásica/patología , Células Cultivadas , Epitelio/patología , Fibroblastos/patología , Proteínas Hedgehog/efectos de los fármacos , Proteínas Hedgehog/genética , Xenoinjertos , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Morfolinas/farmacología , Purinas/farmacología , Quinazolinas/farmacología , Células del Estroma/patología
12.
Proc Natl Acad Sci U S A ; 107(32): 14134-9, 2010 Aug 10.
Artículo en Inglés | MEDLINE | ID: mdl-20679199

RESUMEN

Translational regulation plays a critical role in the control of cell growth and proliferation. A key player in translational control is eIF4E, the mRNA 5' cap-binding protein. Aberrant expression of eIF4E promotes tumorigenesis and has been implicated in cancer development and progression. The activity of eIF4E is dysregulated in cancer. Regulation of eIF4E is partly achieved through phosphorylation. However, the physiological significance of eIF4E phosphorylation in mammals is not clear. Here, we show that knock-in mice expressing a nonphosphorylatable form of eIF4E are resistant to tumorigenesis in a prostate cancer model. By using a genome-wide analysis of translated mRNAs, we show that the phosphorylation of eIF4E is required for translational up-regulation of several proteins implicated in tumorigenesis. Accordingly, increased phospho-eIF4E levels correlate with disease progression in patients with prostate cancer. Our findings establish eIF4E phosphorylation as a critical event in tumorigenesis. These findings raise the possibility that chemical compounds that prevent the phosphorylation of eIF4E could act as anticancer drugs.


Asunto(s)
Factor 4E Eucariótico de Iniciación/metabolismo , Neoplasias/etiología , Neoplasias/patología , Animales , Progresión de la Enfermedad , Factor 4E Eucariótico de Iniciación/genética , Regulación de la Expresión Génica , Técnicas de Sustitución del Gen , Humanos , Masculino , Ratones , Proteínas de Neoplasias/genética , Neoplasias/genética , Fosforilación/fisiología , Regulación hacia Arriba
13.
Nat Commun ; 14(1): 1591, 2023 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-36949044

RESUMEN

In heterogeneous head and neck cancer (HNC), subtype-specific treatment regimens are currently missing. An integrated analysis of patient HNC subtypes using single-cell sequencing and proteome profiles reveals an epithelial-mesenchymal transition (EMT) signature within the epithelial cancer-cell population. The EMT signature coincides with PI3K/mTOR inactivation in the mesenchymal subtype. Conversely, the signature is suppressed in epithelial cells of the basal subtype which exhibits hyperactive PI3K/mTOR signalling. We further identify YBX1 phosphorylation, downstream of the PI3K/mTOR pathway, restraining basal-like cancer cell proliferation. In contrast, YBX1 acts as a safeguard against the proliferation-to-invasion switch in mesenchymal-like epithelial cancer cells, and its loss accentuates partial-EMT and in vivo invasion. Interestingly, phospho-YBX1 that is mutually exclusive to partial-EMT, emerges as a prognostic marker for overall patient outcomes. These findings create a unique opportunity to sensitise mesenchymal cancer cells to PI3K/mTOR inhibitors by shifting them towards a basal-like subtype as a promising therapeutic approach against HNC.


Asunto(s)
Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Humanos , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Proliferación Celular/genética , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal/genética , Línea Celular Tumoral , Movimiento Celular , Proteína 1 de Unión a la Caja Y/genética , Proteína 1 de Unión a la Caja Y/metabolismo
14.
Biochim Biophys Acta Mol Cell Res ; 1869(1): 119140, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34599983

RESUMEN

Translation of an mRNA represents a critical step during the expression of protein-coding genes. As mechanisms governing post-transcriptional regulation of gene expression are progressively unveiled, it is becoming apparent that transcriptional programs are not fully reflected in the proteome. Herein, we highlight a previously underappreciated post-transcriptional mode of regulation of gene expression termed translational buffering. In principle, translational buffering opposes the impact of alterations in mRNA levels on the proteome. We further describe three types of translational buffering: compensation, which maintains protein levels e.g. across species or individuals; equilibration, which retains pathway stoichiometry; and offsetting, which acts as a reversible mechanism that maintains the levels of selected subsets of proteins constant despite genetic alteration and/or stress-induced changes in corresponding mRNA levels. While mechanisms underlying compensation and equilibration have been reviewed elsewhere, the principal focus of this review is on the less-well understood mechanism of translational offsetting. Finally, we discuss potential roles of translational buffering in homeostasis and disease.


Asunto(s)
Homeostasis , Biosíntesis de Proteínas , Animales , Uso de Codones , Humanos , Procesamiento Postranscripcional del ARN , Proteínas Ribosómicas/metabolismo
15.
Front Med (Lausanne) ; 9: 1059122, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36465905

RESUMEN

Background: [177Lu]Lu-PSMA is a radioligand therapy used in metastatic castration-resistant prostate cancer (mCRPC). Despite a survival benefit, the responses for many patients receiving [177Lu]Lu-PSMA are not durable, and all patients eventually develop progressive disease. The bone marrow is the most common site of progression. Micrometastases in this area likely receive an inadequate dose of radiation, as the emitted beta-particles from 177Lu travel an average range of 0.7 mm in soft tissue, well beyond the diameter of micrometastases. Radium-223 (223Ra) is a calcium-mimetic and alpha-emitting radionuclide approved for use in men with mCRPC with bone metastases. The range of emitted alpha particles in soft tissue is much shorter (≤100 µm) with high linear energy transfer, likely more lethal for osseous micrometastases. We anticipate that combining a bone-specific alpha-emitter with [177Lu]Lu-PSMA will improve eradication of micrometastatic osseous disease, and thereby lead to higher and longer responses. Methods: This is a single-center, single-arm phase I/II trial evaluating the combination of 223Ra and [177Lu]Lu-PSMA-I&T in men with mCRPC. Thirty-six patients will receive 7.4 GBq of [177Lu]Lu-PSMA-I&T, concurrently with 223Ra in escalating doses (28 kBq/kg - 55kBq/kg), both given intravenously every six weeks for up to six cycles. Eligible patients will have at least two untreated bone metastases visible on bone scintigraphy, and PSMA-positive disease on PSMA PET scan. Patients must have adequate bone marrow and organ function and be willing to undergo tumor biopsies. Patients with discordant disease visible on FDG PET scan (defined as FDG positive disease with minimal or no PSMA expression and no uptake on bone scan) will be excluded. Other key exclusion criteria include the presence of diffuse marrow disease, prior treatment with 223Ra or [177Lu]Lu-PSMA, or more than one prior line of chemotherapy for prostate cancer. The co-primary objectives of this study are to determine the maximum tolerated dose of 223Ra when combined with [177Lu]Lu-PSMA-I&T and the 50% PSA response rate. Conclusion: The AlphaBet trial is a phase I/II study combining 223Ra with [177Lu]Lu-PSMA-I&T in patients with mCRPC. We aim to enroll the first patient in Q3 2022, and recruitment is anticipated to continue for 24 months. Study registration: NCT05383079.

16.
Cancers (Basel) ; 13(13)2021 Jun 29.
Artículo en Inglés | MEDLINE | ID: mdl-34209750

RESUMEN

Breast and prostate cancer are the second and third leading causes of death amongst all cancer types, respectively. Pathogenesis of these malignancies is characterised by dysregulation of sex hormone signalling pathways, mediated by the estrogen receptor-α (ER) in breast cancer and androgen receptor (AR) in prostate cancer. ER and AR are transcription factors whose aberrant function drives oncogenic transcriptional programs to promote cancer growth and progression. While ER/AR are known to stimulate cell growth and survival by modulating gene transcription, emerging findings indicate that their effects in neoplasia are also mediated by dysregulation of protein synthesis (i.e., mRNA translation). This suggests that ER/AR can coordinately perturb both transcriptional and translational programs, resulting in the establishment of proteomes that promote malignancy. In this review, we will discuss relatively understudied aspects of ER and AR activity in regulating protein synthesis as well as the potential of targeting mRNA translation in breast and prostate cancer.

17.
Cancers (Basel) ; 13(6)2021 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-33799802

RESUMEN

Mast cells (MCs) are important cellular components of the tumor microenvironment and are significantly associated with poor patient outcomes in prostate cancer and other solid cancers. The promotion of tumor progression partly involves heterotypic interactions between MCs and cancer-associated fibroblasts (CAFs), which combine to potentiate a pro-tumor extracellular matrix and promote epithelial cell invasion and migration. Thus far, the interactions between MCs and CAFs remain poorly understood. To identify molecular changes that may alter resident MC function in the prostate tumor microenvironment, we profiled the transcriptome of human prostate MCs isolated from patient-matched non-tumor and tumor-associated regions of fresh radical prostatectomy tissue. Transcriptomic profiling revealed a distinct gene expression profile of MCs isolated from prostate tumor regions, including the downregulation of SAMD14, a putative tumor suppressor gene. Proteomic profiling revealed that overexpression of SAMD14 in HMC-1 altered the secretion of proteins associated with immune regulation and extracellular matrix processes. To assess MC biological function within a model of the prostate tumor microenvironment, HMC-1-SAMD14+ conditioned media was added to co-cultures of primary prostatic CAFs and prostate epithelium. HMC-1-SAMD14+ secretions were shown to reduce the deposition and alignment of matrix produced by CAFs and suppress pro-tumorigenic prostate epithelial morphology. Overall, our data present the first profile of human MCs derived from prostate cancer patient specimens and identifies MC-derived SAMD14 as an important mediator of MC phenotype and function within the prostate tumor microenvironment.

18.
Mol Cancer Ther ; 20(11): 2140-2150, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34413130

RESUMEN

Monotherapy with PARP inhibitors is effective for the subset of castrate-resistant prostate cancer (CRPC) with defects in homologous recombination (HR) DNA repair. New treatments are required for the remaining tumors, and an emerging strategy is to combine PARP inhibitors with other therapies that induce DNA damage. Here we tested whether PARP inhibitors are effective for HR-proficient CRPC, including androgen receptor (AR)-null tumors, when used in combination with CX-5461, a small molecule that inhibits RNA polymerase I transcription and activates the DNA damage response, and has antitumor activity in early phase I trials. The combination of CX-5461 and talazoparib significantly decreased in vivo growth of patient-derived xenografts of HR-proficient CRPC, including AR-positive, AR-null, and neuroendocrine tumors. CX-5461 and talazoparib synergistically inhibited the growth of organoids and cell lines, and significantly increased the levels of DNA damage. Decreased tumor growth after combination therapy was maintained for 2 weeks without treatment, significantly increasing host survival. Therefore, combination treatment with CX-5461 and talazoparib is effective for HR-proficient tumors that are not suitable for monotherapy with PARP inhibitors, including AR-null CRPC. This expands the spectrum of CRPC that is sensitive to PARP inhibition.


Asunto(s)
Benzotiazoles/uso terapéutico , Daño del ADN/genética , Naftiridinas/uso terapéutico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Animales , Benzotiazoles/farmacología , Humanos , Masculino , Ratones , Naftiridinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología
19.
Nat Commun ; 12(1): 5049, 2021 08 19.
Artículo en Inglés | MEDLINE | ID: mdl-34413304

RESUMEN

Preclinical testing is a crucial step in evaluating cancer therapeutics. We aimed to establish a significant resource of patient-derived xenografts (PDXs) of prostate cancer for rapid and systematic evaluation of candidate therapies. The PDX collection comprises 59 tumors collected from 30 patients between 2012-2020, coinciding with availability of abiraterone and enzalutamide. The PDXs represent the clinico-pathological and genomic spectrum of prostate cancer, from treatment-naïve primary tumors to castration-resistant metastases. Inter- and intra-tumor heterogeneity in adenocarcinoma and neuroendocrine phenotypes is evident from bulk and single-cell RNA sequencing data. Organoids can be cultured from PDXs, providing further capabilities for preclinical studies. Using a 1 x 1 x 1 design, we rapidly identify tumors with exceptional responses to combination treatments. To govern the distribution of PDXs, we formed the Melbourne Urological Research Alliance (MURAL). This PDX collection is a substantial resource, expanding the capacity to test and prioritize effective treatments for prospective clinical trials in prostate cancer.


Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Organoides/patología , Neoplasias de la Próstata/patología , Animales , Modelos Animales de Enfermedad , Genoma , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mutación , Metástasis de la Neoplasia , Organoides/metabolismo , Estudios Prospectivos , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Bancos de Tejidos , Transcriptoma , Ensayos Antitumor por Modelo de Xenoinjerto
20.
RNA ; 14(2): 324-35, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18094122

RESUMEN

Messenger RNAs are associated with multiple RNA-binding proteins to form ribonucleoprotein (mRNP) complexes. These proteins are important regulators of the fate of their target mRNAs. In human cells, Staufen1 and Staufen2 proteins, coded by two different genes, are double-stranded RNA-binding proteins involved in several cellular functions including mRNA localization, translation, and decay. Although 51% identical, these proteins are nevertheless found in different RNA particles. In addition, differential splicing events generate Staufen2 isoforms that only differ at their N-terminal extremities. In this paper, we used a genome-wide approach to identify and compare the mRNA targets of mammalian Staufen proteins. The mRNA content of Staufen mRNPs was identified by probing DNA microarrays with probes derived from mRNAs isolated from immunopurified Staufen-containing complexes following transfection of HEK293T cells with Stau1(55)-HA, Stau2(59)-HA, or Stau2(62)-HA expressors. Our results indicate that 7% and 11% of the cellular RNAs expressed in HEK293T cells are found in Stau1- and in Stau2-containing mRNPs, respectively. A comparison of Stau1- and Stau2-containing mRNAs identifies a relatively low percentage of common mRNAs; the percentage of common mRNAs highly increases when mRNAs in Stau2(59)-HA- and Stau2(62)-containing mRNPs are compared. There is a predominance of mRNAs involved in cell metabolism, transport, transcription, regulation of cell processes, and catalytic activity. All these subsets of mRNAs are mostly distinct from those associated with FMRP or IMP, although some mRNAs overlap. Consistent with a model of post-transcriptional gene regulation, our results show that Stau1- and Stau2-mRNPs associate with distinct but overlapping sets of cellular mRNAs.


Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Proteínas del Tejido Nervioso/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Secuencia de Aminoácidos , Línea Celular , Proteínas del Citoesqueleto/análisis , Proteínas del Citoesqueleto/genética , Biblioteca de Genes , Genoma Humano , Humanos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/genética , ARN Mensajero/análisis , Proteínas de Unión al ARN/análisis , Proteínas de Unión al ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
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