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1.
bioRxiv ; 2023 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-37503050

RESUMO

p16 is a tumor suppressor encoded by the CDKN2A gene whose expression is lost in ~50% of all human cancers. In its canonical role, p16 inhibits the G1-S phase cell cycle progression through suppression of cyclin dependent kinases. Interestingly, p16 also has roles in metabolic reprogramming, and we previously published that loss of p16 promotes nucleotide synthesis via the pentose phosphate pathway. Whether other nucleotide metabolic genes and pathways are affected by p16/CDKN2A loss and if these can be specifically targeted in p16/CDKN2A-low tumors has not been previously explored. Using CRISPR KO libraries in multiple isogenic human and mouse melanoma cell lines, we determined that many nucleotide metabolism genes are negatively enriched in p16/CDKN2A knockdown cells compared to controls. Indeed, many of the genes that are required for survival in the context of low p16/CDKN2A expression based on our CRISPR screens are upregulated in p16 knockdown melanoma cells and those with endogenously low CDKN2A expression. We determined that cells with low p16/Cdkn2a expression are sensitive to multiple inhibitors of de novo purine synthesis, including anti-folates. Tumors with p16 knockdown were more sensitive to the anti-folate methotrexate in vivo than control tumors. Together, our data provide evidence to reevaluate the utility of these drugs in patients with p16/CDKN2A-low tumors as loss of p16/CDKN2A may provide a therapeutic window for these agents.

2.
Mol Hum Reprod ; 25(3): 124-136, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30590698

RESUMO

Molecular mechanisms responsible for the initiation of primate spermatogenesis remain poorly characterized. Previously, 48 h stimulation of the testes of three juvenile rhesus monkeys with pulsatile LH and FSH resulted in down-regulation of a cohort of genes recognized to favor spermatogonia stem cell renewal. This change in genetic landscape occurred in concert with amplification of Sertoli cell proliferation and the commitment of undifferentiated spermatogonia to differentiate. In this report, the non-protein coding small RNA transcriptomes of the same testes were characterized using RNA sequencing: 537 mature micro-RNAs (miRNAs), 322 small nucleolar RNAs (snoRNAs) and 49 small nuclear RNAs (snRNAs) were identified. Pathway analysis of the 20 most highly expressed miRNAs suggested that these transcripts contribute to limiting the proliferation of the primate Sertoli cell during juvenile development. Gonadotrophin treatment resulted in differential expression of 35 miRNAs, 12 snoRNAs and four snRNA transcripts. Ten differentially expressed miRNAs were derived from the imprinted delta-like homolog 1-iodothyronine deiodinase 3 (DLK1-DIO3) locus that is linked to stem cell fate decisions. Four gonadotrophin-regulated expressed miRNAs were predicted to trigger a local increase in thyroid hormone activity within the juvenile testis. The latter finding leads us to predict that, in primates, a gonadotrophin-induced selective increase in testicular thyroid hormone activity, together with the established increase in androgen levels, at the onset of puberty is necessary for the normal timing of Sertoli cell maturation, and therefore initiation of spermatogenesis. Further examination of this hypothesis requires that peripubertal changes in thyroid hormone activity of the testis of a representative higher primate be determined empirically.


Assuntos
MicroRNAs/metabolismo , Testículo/metabolismo , Hormônios Tireóideos/metabolismo , Animais , Hormônio Foliculoestimulante/metabolismo , Hormônio Luteinizante/metabolismo , Macaca mulatta , Masculino , MicroRNAs/genética , Análise de Sequência de RNA , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Espermatogênese/genética , Espermatogênese/fisiologia , Transcriptoma/genética
3.
Int J Cancer ; 143(6): 1388-1401, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-29633255

RESUMO

Our recent studies have shown that cross-talk between histone deacetylase 5 (HDAC5) and lysine-specific demethylase 1 (LSD1) facilitates breast cancer progression. In this work, we demonstrated that regulatory activity at -356 to -100 bp promoter element plays a critical role in governing HDAC5 transcription. By using DNA affinity precipitation and mass spectrometry, we identified a group of factors that bind to this element. Among these factors, Upstream Transcription Factor 1 (USF1) was shown to play a critical role in controlling HDAC5 transcription. Through screening a panel of epigenetic modifying drugs, we showed that a natural bioactive HDAC inhibitor, sulforaphane, downregulated HDAC5 transcription by blocking USF1 activity. Sulforaphane facilitated LSD1 ubiquitination and degradation in an HDAC5-dependent manner. A comparative microarray analysis demonstrated a genome wide cooperative effect of HDAC5 and LSD1 on cancer-related gene expression. shRNA knockdown and sulforaphane inhibition of HDAC5/LSD1 exhibited similar effects on expression of HDAC5/LSD1 target genes. We also showed that coordinated cross-talk of HDAC5 and LSD1 is essential for the antitumor efficacy of sulforaphane. Combination treatment with sulforaphane and a potent LSD1 inhibitor resulted in synergistic growth inhibition in breast cancer cells, but not in normal breast epithelial cells. Furthermore, combined therapy with sulforaphane and LSD1 inhibitor exhibited superior inhibitory effect on MDA-MB-231 xenograft tumor growth. Taken together, our work demonstrates that HDAC5-LSD1 axis is an effective drug target for breast cancer. Inhibition of HDAC5-LSD1 axis with sulforaphane blocks breast cancer growth and combined treatment with LSD1 inhibitor improves the therapeutic efficacy of sulforaphane.


Assuntos
Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona Desacetilases/metabolismo , Histona Desmetilases/metabolismo , Isotiocianatos/farmacologia , Animais , Biomarcadores Tumorais/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Proliferação de Células/efeitos dos fármacos , Epigênese Genética , Feminino , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/química , Histona Desacetilases/genética , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , RNA Interferente Pequeno/genética , Sulfóxidos , Células Tumorais Cultivadas , Fatores Estimuladores Upstream/genética , Fatores Estimuladores Upstream/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
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