RESUMO
General control nonderepressible 2 (GCN2) protein kinase is a cellular stress sensor within the tumor microenvironment (TME), whose signaling cascade has been proposed to contribute to immune escape in tumors. Herein, we report the discovery of cell-potent GCN2 inhibitors with excellent selectivity against its closely related Integrated Stress Response (ISR) family members heme-regulated inhibitor kinase (HRI), protein kinase R (PKR), and (PKR)-like endoplasmic reticulum kinase (PERK), as well as good kinome-wide selectivity and favorable PK. In mice, compound 39 engages GCN2 at levels ≥80% with an oral dose of 15 mg/kg BID. We also demonstrate the ability of compound 39 to alleviate MDSC-related T cell suppression and restore T cell proliferation, similar to the effect seen in MDSCs from GCN2 knockout mice. In the LL2 syngeneic mouse model, compound 39 demonstrates significant tumor growth inhibition (TGI) as a single agent. Furthermore, TGI mediated by anti-VEGFR was enhanced by treatment with compound 39 demonstrating the complementarity of these two mechanisms.
Assuntos
Células Supressoras Mieloides , eIF-2 Quinase , Animais , Heme , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases , Linfócitos T/metabolismo , eIF-2 Quinase/metabolismoRESUMO
Recent studies have shown that microRNA (miRNA) regulates gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Here, we report new evidence in which miRNA may also function to induce gene expression. By scanning gene promoters in silico for sequences complementary to known miRNAs, we identified a putative miR-373 target site in the promoter of E-cadherin. Transfection of miR-373 and its precursor hairpin RNA (pre-miR-373) into PC-3 cells readily induced E-cadherin expression. Knockdown experiments confirmed that induction of E-cadherin by pre-miR-373 required the miRNA maturation protein Dicer. Further analysis revealed that cold-shock domain-containing protein C2 (CSDC2), which possesses a putative miR-373 target site within its promoter, was also readily induced in response to miR-373 and pre-miR-373. Furthermore, enrichment of RNA polymerase II was detected at both E-cadherin and CSDC2 promoters after miR-373 transfection. Mismatch mutations to miR-373 indicated that gene induction was specific to the miR-373 sequence. Transfection of promoter-specific dsRNAs revealed that the concurrent induction of E-cadherin and CSDC2 by miR-373 required the miRNA target sites in both promoters. In conclusion, we have identified a miRNA that targets promoter sequences and induces gene expression. These findings reveal a new mode by which miRNAs may regulate gene expression.
Assuntos
Regulação da Expressão Gênica , MicroRNAs/fisiologia , Ativação Transcricional , Pareamento Incorreto de Bases , Sequência de Bases , Caderinas/genética , Linhagem Celular Tumoral , Expressão Gênica/efeitos dos fármacos , Humanos , MicroRNAs/genética , MicroRNAs/farmacologia , Dados de Sequência Molecular , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Polimerase II/metabolismo , Proteínas de Ligação a RNA/genética , Ribonuclease III/antagonistas & inibidores , Ribonuclease III/genética , Ribonuclease III/metabolismoRESUMO
BACKGROUND: Checkpoint inhibitors (CPIs) such as anti-PD(L)-1 and anti-CTLA-4 antibodies have resulted in unprecedented rates of antitumor responses and extension of survival of patients with a variety of cancers. But some patients fail to respond or initially respond but later relapse as they develop resistance to immune therapy. One of the tumor-extrinsic mechanisms for resistance to immune therapy is the accumulation of regulatory T cells (Treg) in tumors. In preclinical and clinical studies, it has been suggested that tumor trafficking of Treg is mediated by CC chemokine receptor 4 (CCR4). Over 90% of human Treg express CCR4 and migrate toward CCL17 and CCL22, two major CCR4 ligands that are either high at baseline or upregulated in tumors on CPI treatment. Hence, CCR4 antagonism has the potential to be an effective antitumor treatment by reducing the accumulation of Treg into the tumor microenvironment (TME). METHODS: We developed in vitro and in vivo models to assess Treg migration and antitumor efficacy using a potent and selective CCR4 antagonist, CCR4-351. We used two separate tumor models, Pan02 and CT26 mouse tumors, that have high and low CCR4 ligand expression, respectively. Tumor growth inhibition as well as the frequency of tumor-infiltrating Treg and effector T cells was assessed following the treatment with CCR4 antagonist alone or in combination with CPI. RESULTS: Using a selective and highly potent, novel small molecule inhibitor of CCR4, we demonstrate that migration of CCR4+ Treg into the tumor drives tumor progression and resistance to CPI treatment. In tumor models with high baseline levels of CCR4 ligands, blockade of CCR4 reduced the number of Treg and enhanced antitumor immune activity. Notably, in tumor models with low baseline level of CCR4 ligands, treatment with immune CPIs resulted in significant increases of CCR4 ligands and Treg numbers. Inhibition of CCR4 reduced Treg frequency and potentiated the antitumor effects of CPIs. CONCLUSION: Taken together, we demonstrate that CCR4-dependent Treg recruitment into the tumor is an important tumor-extrinsic mechanism for immune resistance. Blockade of CCR4 led to reduced frequency of Treg and resulted in increased antitumor activity, supporting the clinical development of CCR4 inhibitors in combination with CPI for the treatment of cancer. STATEMENT OF SIGNIFICANCE: CPI upregulates CCL17 and CCL22 expression in tumors and increases Treg migration into the TME. Pharmacological antagonism of the CCR4 receptor effectively inhibits Treg recruitment and results in enhanced antitumor efficacy either as single agent in CCR4 ligandhigh tumors or in combination with CPIs in CCR4 ligandlow tumors.
Assuntos
Imunoterapia/métodos , Neoplasias/imunologia , Neoplasias/terapia , Receptores CCR4/imunologia , Linfócitos T Reguladores/imunologia , Animais , Feminino , Humanos , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The deubiquitinase USP7 regulates the levels of multiple proteins with roles in cancer progression and immune response. Thus, USP7 inhibition may decrease oncogene function, increase tumor suppressor function, and sensitize tumors to DNA-damaging agents. We have discovered a novel chemical series that potently and selectively inhibits USP7 in biochemical and cellular assays. Our inhibitors reduce the viability of multiple TP53 wild-type cell lines, including several hematologic cancer and MYCN-amplified neuroblastoma cell lines, as well as a subset of TP53-mutant cell lines in vitro Our work suggests that USP7 inhibitors upregulate transcription of genes normally silenced by the epigenetic repressor complex, polycomb repressive complex 2 (PRC2), and potentiate the activity of PIM and PI3K inhibitors as well as DNA-damaging agents. Furthermore, oral administration of USP7 inhibitors inhibits MM.1S (multiple myeloma; TP53 wild type) and H526 (small cell lung cancer; TP53 mutant) tumor growth in vivo Our work confirms that USP7 is a promising, pharmacologically tractable target for the treatment of cancer.
Assuntos
Peptidase 7 Específica de Ubiquitina/antagonistas & inibidores , Animais , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Modelos MolecularesRESUMO
USP7 is a promising target for cancer therapy as its inhibition is expected to decrease function of oncogenes, increase tumor suppressor function, and enhance immune function. Using a structure-based drug design strategy, a new class of reversible USP7 inhibitors has been identified that is highly potent in biochemical and cellular assays and extremely selective for USP7 over other deubiquitinases. The succinimide was identified as a key potency-driving motif, forming two strong hydrogen bonds to the allosteric pocket of USP7. Redesign of an initial benzofuran-amide scaffold yielded a simplified ether series of inhibitors, utilizing acyclic conformational control to achieve proper amine placement. Further improvements were realized upon replacing the ether-linked amines with carbon-linked morpholines, a modification motivated by free energy perturbation (FEP+) calculations. This led to the discovery of compound 41, a highly potent, selective, and orally bioavailable USP7 inhibitor. In xenograft studies, compound 41 demonstrated tumor growth inhibition in both p53 wildtype and p53 mutant cancer cell lines, demonstrating that USP7 inhibitors can suppress tumor growth through multiple different pathways.
Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/química , Descoberta de Drogas/métodos , Peptidase 7 Específica de Ubiquitina/antagonistas & inibidores , Peptidase 7 Específica de Ubiquitina/química , Administração Oral , Animais , Linhagem Celular Tumoral , Cristalografia por Raios X/métodos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Estrutura Terciária de Proteína , Peptidase 7 Específica de Ubiquitina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Small double stranded RNAs (dsRNA) are a new class of molecules which regulate gene expression. Accumulating data suggest that some dsRNA can function as tumor suppressors. Here, we report further evidence on the potential of dsRNA mediated p21 induction. Using the human renal cell carcinoma cell line A498, we found that dsRNA targeting the p21 promoter significantly induced the expression of p21 mRNA and protein levels. As a result, dsP21 transfected cells had a significant decrease in cell viability with a concomitant G1 arrest. We also observed a significant increase in apoptosis. These findings were associated with a significant decrease in survivin mRNA and protein levels. This is the first report that demonstrates dsRNA mediated gene activation in renal cell carcinoma and suggests that forced over-expression of p21 may lead to an increase in apoptosis through a survivin dependent mechanism.
Assuntos
Apoptose/genética , Carcinoma de Células Renais/patologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Fase G1/genética , Neoplasias Renais/patologia , RNA de Cadeia Dupla/fisiologia , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Primers do DNA , Citometria de Fluxo , Humanos , Proteínas Inibidoras de Apoptose , Proteínas Associadas aos Microtúbulos/genética , RNA Mensageiro/genética , SurvivinaRESUMO
Androgen receptor (AR) is a ligand-activated transcription factor belonging to the steroid hormone receptor family and is very important for the development and progression of prostate cancer. The soy isoflavone genistein has been shown previously to down-regulate AR in androgen-dependent prostate cancer cell lines such as LNCaP. However, the mechanism(s) by which AR is down-regulated by genistein is still not known fully. We show a new mechanism by which genistein inhibits AR protein levels. We show that genistein-treated LNCaP cells exhibit increased ubiquitination of AR, suggesting that AR protein is down-regulated via a proteasome-mediated pathway. AR is normally stabilized by the chaperone activity of the heat shock protein Hsp90. The increased ubiquitination of AR after genistein treatment is attributed to decreased Hsp90 chaperone activity as assessed by its increased functionally inactive acetylated form. Consistent with this result, we find that HDAC6, which is a Hsp90 deacetylase, is inhibited by the antiestrogenic activity of genistein. Hence, in this study, we elucidate a novel mechanism of AR down-regulation by genistein through inhibition of HDAC6-Hsp90 cochaperone function required to stabilize AR protein. Our results suggest that genistein could be used as a potential chemopreventive agent for prostate cancers along with known inhibitors of HDAC6 and Hsp90.
Assuntos
Antineoplásicos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Genisteína/farmacologia , Proteínas de Choque Térmico HSP90/metabolismo , Histona Desacetilases/metabolismo , Receptores Androgênicos/metabolismo , Antagonistas de Receptores de Andrógenos , Estradiol/farmacologia , Desacetilase 6 de Histona , Humanos , Masculino , Transporte Proteico/efeitos dos fármacos , TermodinâmicaRESUMO
We recently reported that synthetic dsRNAs targeting promoter regions can induce gene expression in a phenomenon referred to as dsRNA-induced gene activation/RNA activation (RNAa) [Li et al. Proc Natl Acad Sci U S A 2006;103:17337-42]. The present study investigates the in vitro antitumor activity RNAa can elicit through triggering the expression of cell cycle repressor protein p21(WAF1/CIP1) (p21) in human bladder cancer cells. Transfection of a 21-nucleotide dsRNA targeting the p21 promoter (dsP21) was used to induce p21 expression in T24 and J82 bladder cancer cell lines. Reverse transcription-PCR and Western blot analysis accessed the increase p21 mRNA and protein levels, respectively, in transfected cells. In association to p21 induction, dsP21 transfection significantly inhibited bladder cancer cell proliferation and clonogenicity. Further analysis of cell viability and cell cycle distribution revealed that dsP21 transfection also enhanced apoptotic cell death and caused an accumulation in the G(1) phase in both cell lines. In conclusion, p21 activation by RNAa has antitumor activity in vitro in bladder cancer cells. These results suggest that RNAa could be used for cancer treatment by targeted activation of tumor suppressor genes.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , RNA de Cadeia Dupla/fisiologia , Neoplasias da Bexiga Urinária/genética , Western Blotting , Linhagem Celular Tumoral , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Neoplasias da Bexiga Urinária/enzimologia , Neoplasias da Bexiga Urinária/patologiaRESUMO
Recruitment of suppressive CD4+ FOXP3+ regulatory T cells (Treg) to the tumor microenvironment (TME) has the potential to weaken the antitumor response in patients receiving treatment with immuno-oncology (IO) agents. Human Treg express CCR4 and can be recruited to the TME through the CC chemokine ligands CCL17 and CCL22. In some cancers, Treg accumulation correlates with poor patient prognosis. Preclinical data suggests that preventing the recruitment of Treg and increasing the population of activated effector T cells (Teff) in the TME can potentiate antitumor immune responses. We developed a novel series of potent, orally bioavailable small molecule antagonists of CCR4. From this series, several compounds exhibited high potency in distinct functional assays in addition to good in vitro and in vivo ADME properties. The design, synthesis, and SAR of this series and confirmation of its in vivo activity are reported.
Assuntos
Movimento Celular/efeitos dos fármacos , Pirazinas/farmacologia , Pirazóis/farmacologia , Receptores CCR4/antagonistas & inibidores , Linfócitos T Reguladores/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Animais , Cicloexanos/síntese química , Cicloexanos/farmacocinética , Cicloexanos/farmacologia , Descoberta de Drogas , Humanos , Camundongos Transgênicos , Estrutura Molecular , Piperazinas/síntese química , Piperazinas/farmacocinética , Piperazinas/farmacologia , Pirazinas/síntese química , Pirazinas/farmacocinética , Pirazóis/síntese química , Pirazóis/farmacocinética , Ratos , Relação Estrutura-AtividadeRESUMO
PURPOSE: The RAS-association domain family 1, isoform A (RASSF1A) gene is shown to be inactivated in prostate cancers. However, the molecular mechanism of silencing of the RASSFIA gene is not fully understood. The present study was designed to investigate the mechanisms of inactivation of the RASSF1A gene through the analysis of CpG methylation and histone acetylation and H3 methylation associated with the RASSF1A promoter region. EXPERIMENTAL DESIGN: Methylation status of the RASSF1A gene was analyzed in 131 samples of prostate cancer, 65 samples of benign prostate hypertrophy (BPH), and human prostate cell lines using methylation-specific PCR. Histone acetylation (acetyl-H3, acetyl-H4) and H3 methylation (dimethyl-H3-K4, dimethyl-H3-K9) status associated with the promoter region in prostate cells were analyzed by chromatin immunoprecipitation (ChIP) assay. RESULTS: Aberrant methylation was detected in 97 (74.0%) prostate cancer samples and 12 (18.5%) BPH samples. The methylation frequency of RASSF1A showed a significant increase with high Gleason sum and high stage. The ChIP assays showed enhancement of histone acetylation and dimethyl-H3-K4 methylation on the unmethylated RASSF1A promoter. TSA alone was unable to alter key components of the histone code. However, after 5-aza-2'-deoxy-cytidine treatment, there was a complete reversal of the histone components in the hypermethylated promoter. Levels of acetyl-H3, acetyl-H4, and dimethyl-H3-K4 became more enriched, whereas H3K9me2 levels were severely depleted. CONCLUSIONS: This is the first report suggesting that reduced histone acetylation or H3K4me2 methylation and increased dimethyl-H3-K9 methylation play a critical role in the maintenance of promoter DNA methylation-associated RASSF1A gene silencing in prostate cancer.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/genética , Proteínas Supressoras de Tumor/genética , Acetilação , Idoso , Idoso de 80 Anos ou mais , Metilação de DNA , Epigênese Genética , Histonas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/metabolismoRESUMO
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD; dioxin) is a toxic environmental contaminant that works through dioxin response elements (DRE) to activate gene expression. We tested the hypothesis that cancer-related epigenetic changes suppress dioxin activation of the cytochrome P4501A1 (CYP1A1) gene. 5-Aza-2'-deoxycytidine (5-aza-CdR), an inhibitor of DNA methylation, increases TCDD-inducible CYP1A1 mRNA expression in cancerous LNCaP cells but not in noncancerous PWR-1E and RWPE-1 cells (all human prostate cell lines). Bisulfite DNA sequencing shows that the TCDD-responsive CYP1A1 enhancer is highly methylated in LNCaP cells but not in RWPE-1 cells. In vivo footprinting experiments reveal that unmethylated DRE sites do not bind protein in response to TCDD in LNCaP cells, whereas inducible DRE occupancy occurs in RWPE-1 cells. Pretreatment of LNCaP cells with 5-aza-CdR partially restores TCDD-inducible DRE occupancy, showing that DNA methylation indirectly suppresses DRE occupancy. Chromatin immunoprecipitation experiments reveal that LNCaP cells lack trimethyl histone H3 lysine 4, a mark of active genes, on the CYP1A1 regulatory region, whereas this histone modification is prevalent in PWR-1E and RWPE-1 cells. We also analyzed CYP1A1 enhancer methylation in human prostate tissue DNA. We do not detect CYP1A1 enhancer methylation in 30 DNA samples isolated from noncancerous prostate tissue. In contrast, 11 of 30 prostate tumor DNA samples have detectable CYP1A1 enhancer methylation, indicating that it is hypermethylated in prostate tumors. This is the first report that shows that CYP1A1 is aberrantly hypermethylated in human prostate cancer and has an altered, inaccessible chromatin structure that suppresses its dioxin responsiveness.
Assuntos
Citocromo P-450 CYP1A1/genética , Dioxinas/farmacologia , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Ilhas de CpG , Citocromo P-450 CYP1A1/biossíntese , Metilação de DNA/efeitos dos fármacos , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Decitabina , Elementos Facilitadores Genéticos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , Masculino , Elementos de RespostaRESUMO
The human CYP1A genes CYP1A1 and CYP1A2 are in a head-to-head orientation on chromosome 15. Both CYP1A genes and CYP1B1 are transcriptionally induced by the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that binds 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin). Although the TCDD-responsive enhancers for CYP1A1 and CYP1B1 are well characterized, a similar CYP1A2 enhancer has not been identified. In the human prostate cell line RWPE-1, CYP1A2 mRNA expression is dramatically induced by TCDD. Therefore, analysis of the native CYP1A2 gene in these cells can provide insight into its induction mechanism. To identify sites that may bind AhR on the CYP1A locus, we scanned 75 kilobases of chromosome 15 sequence for high-affinity AhR binding sites. We then analyzed most of the sites for TCDD-inducible AhR interaction by chromatin immunoprecipitation. As expected, the CYP1A1 and CYP1B1 enhancers bind AhR in TCDD-treated cells. It is noteworthy that we identify a region 3' of CYP1A2 that also binds AhR in response to TCDD. We cannot detect AhR binding at other sites on the CYP1A locus. In vivo footprinting demonstrates that two AhR binding sites in the CYP1A2 3' region are occupied in TCDD-treated cells. Reporter-gene studies show that these sites confer TCDD-responsiveness to a heterologous promoter. AhR also binds to the CYP1A2 3' region in TCDD-treated LS180 cells but not in HepG2 and ND-1 cells. In the latter cell lines, the CYP1A2 3' region is extensively methylated. In summary, we identify a novel TCDD-responsive enhancer for CYP1A2. We were surprised to find that this enhancer is not conserved across species and is primarily human-specific.
Assuntos
Região 3'-Flanqueadora/genética , Citocromo P-450 CYP1A2/biossíntese , Citocromo P-450 CYP1A2/genética , Elementos Facilitadores Genéticos/fisiologia , Dibenzodioxinas Policloradas/farmacologia , Região 3'-Flanqueadora/efeitos dos fármacos , Adulto , Sequência de Bases , Linhagem Celular Transformada , Linhagem Celular Tumoral , Elementos Facilitadores Genéticos/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência MolecularRESUMO
Various carcinogenic metabolites, including catechol estrogens, play a role in malignant transformation. An enzyme that is capable of neutralizing the genotoxic effects of these compounds is catechol-O-methyltransferase (COMT). A variant form of this enzyme has been shown to reduce its activity by up to 4-fold; thus, we hypothesize that single nucleotide polymorphisms of the COMT gene can be a risk factor for benign prostatic hyperplasia (BPH) and prostate cancer. To test this hypothesis, the genetic distribution of three different COMT polymorphisms at codon 62 (C-->T), codon 72 (G-->T), and codon 158 (G-->A) were analyzed in 131 normal healthy subjects, 134 BPH, and 178 sporadic prostate cancer samples from a Japanese population. Results of these experiments show that the variant genotype at codon 62 (P = 0.060) and codon 158 (P = 0.047) are risk factors for prostate cancer but not BPH when compared with normal controls. Odds ratio (OR) and 95% confidence interval (95% CI) for cancer were 3.24 and 1.38 to 7.61, respectively, for codon 62 T/T genotype when compared with wild type. At codon 158, the A/A variant for cancer had an OR of 3.00 with a 95% CI of 1.38 to 6.54 compared with wild type. Codons 62 and 158 were in linkage disequilibrium (LD), and when compared with the C-G haplotype, other types (C-A, T-G, T-A) were observed to be associated with prostate cancer (P = 0.040) but not BPH. Codon 72 on the other hand, was not in LD with either codon 62 or 158. The homozygous variant on codon 72 was rare in this Japanese population, and the heterozygous G/T at this codon was not associated with either prostate cancer or BPH. When evaluating the risk of COMT polymorphisms with stage or grade of cancer, no associations were observed for any of the genotypes with the exception of a tendency (P = 0.096) for the variant A allele on codon 158 to be correlated with higher stages (> or = T3) of cancer. This is the first report that shows the polymorphisms of COMT to be associated with sporadic prostatic carcinogenesis. These results are important in understanding the role of COMT polymorphisms in the pathogenesis of prostate cancer.
Assuntos
Catecol O-Metiltransferase/genética , Ligação Genética , Polimorfismo Genético , Hiperplasia Prostática/genética , Neoplasias da Próstata/genética , Alelos , Códon , Genótipo , Humanos , Japão , MasculinoRESUMO
beta-Catenin and transcriptional factor TCF-4 (human T-cell factor-4) genes comprise the Wnt signal. The Wnt signal pathway plays an important role in malignant transformation. We hypothesize that the beta-catenin and TCF-4 gene and Wnt signal are important in the progression of renal cell carcinoma (RCC). To test this hypothesis, we investigated TCF-4 splicing isoforms, beta-catenin, and Wnt signal pathway (cyclin D1, c-myc, c-jun, and MMP7) in three RCC cell lines (A498, Caki-1, and Caki-2), 38 primary RCCs, and 29 normal kidney samples. We also analyzed the relationship between TCF-4 gene splicing isoforms, proliferation (proliferating cell nuclear antigen labeling index), and apoptosis [antiapoptotic factors (Bcl-2 and Bcl-x(L)), proapoptotic factors (Bak and Bax), and caspase-3] in RCC samples. In 38 RCC samples, four splicing isoforms of the TCF-4 gene were present in the region between exon 12 and exon 17. Thirty (79%) of 38 RCCs and all (100%) of the normal kidney samples showed mixed isoforms with both long and short reading frames in the COOH-terminal region, whereas the remaining 8 RCC samples showed only the long-form reading frame. Two COOH-terminal-binding protein sites were present only in the long-form reading frame. The eight RCCs that demonstrated only the long reading frame isoform showed early disease progression and poor prognosis. In these 8 RCC samples, down-regulation of cyclin D1, c-myc, c-jun, and MMP7 expression was observed at the mRNA level. In addition, a marked reduction of caspase-3 expression was also found at both the mRNA and the protein level. However, the beta-catenin gene was not overexpressed at the mRNA level and protein level, and mutation and deletion were not observed in exon 3. In these three renal cell lines, there was no significant difference in TCF-4 mRNA expression before and after 5-Aza-2'-deoxycytidine treatment, and there appeared to be no splicing isoforms in the region between exon 1 and exon 11. These findings suggest that alteration in beta-catenin is an infrequent event in RCC. In samples in which beta-catenin was not overexpressed, the target genes of Wnt signal were regulated through TCF-4 splicing isoforms. The imbalance between TCF-4 gene splicing isoforms with long and short reading frames is associated with RCC progression through the inhibition of the apoptotic pathway. We demonstrate for the first time that TCF-4 gene splicing isoforms and the Wnt signal pathway can induce progression of RCC by the inhibition of apoptosis and not by the induction of cell proliferation.
Assuntos
Apoptose , Carcinoma de Células Renais/patologia , Proteínas de Ligação a DNA/genética , Neoplasias Renais/patologia , Proteínas Proto-Oncogênicas/fisiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Sequência de Bases , Divisão Celular , Proteínas do Citoesqueleto/genética , Humanos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Polimorfismo Conformacional de Fita Simples , Prognóstico , Isoformas de Proteínas , RNA Mensageiro/análise , Fatores de Transcrição TCF , Transativadores/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição , Proteínas Wnt , beta CateninaRESUMO
MicroRNAs (miRNAs) are a class of small non-coding RNAs (ncRNAs) that regulate gene expression by repressing translation or triggering the degradation of complementary mRNA sequences. Certain miRNAs have been shown to function as integral components of the p53 and/or retinoblastoma (Rb) regulatory networks. As such, miRNA dysregulation can have a profound effect on cancer development. Previous studies have shown that miR-449a is down-regulated in human prostate cancer tissue and possesses potential tumor suppressor function. In the present study, we identify miR-449a-mediated growth arrest in prostate cancer cells is dependent on the Rb protein. We show that mutant Rb prostate cancer cells (DU- 145) are resistant to cell cycle arrest and cellular senescence induced by miR- 449a, while overexpression of wild-type Rb in DU-145 sublines (DU-1.1 and B5) restores miR-449a function. In silico analysis of 3'UTR regions reveal a putative miR-449a target site in the transcript of Cyclin D1 (CCND1); an oncogene involved in directly regulating Rb activity and cell cycle progression. Luciferase 3'UTR reporter constructs and inhibitory oligonucleotides confirm that Cyclin D1 is a direct downstream target of miR-449a. We also reveal that miR-449a suppresses Rb phosphorylation through the knockdown of Cyclin D1 and previously validated target HDAC1. By targeting genes involved in controlling Rb activity, miR- 449a regulates growth and senescence in an Rb-dependent manner. These data indicate that miR-449a is a miRNA component of the Rb pathway and its tumor suppressor-like effects, in part, depends on Rb status in prostate cancer cells.
Assuntos
Ciclo Celular , Proliferação de Células , Senescência Celular , MicroRNAs/metabolismo , Neoplasias da Próstata/genética , Proteína do Retinoblastoma/metabolismo , Regiões 3' não Traduzidas , Apoptose , Sítios de Ligação , Linhagem Celular Tumoral , Ciclina D1/genética , Ciclina D1/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Humanos , Masculino , Mutação , Fosforilação , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Interferência de RNA , Proteína do Retinoblastoma/genética , TransfecçãoRESUMO
The aryl hydrocarbon receptor (AhR) is a ligand-activated regulatory protein that controls estrogen action through two distinct pathways. In one pathway, AhR acts as a transcription factor that induces the expression of the CYP1 family of estrogen-metabolizing genes; in the other pathway, AhR initiates the degradation of the estrogen receptor and suppresses estrogen signaling. The AhR ligand 3,3'-diindolylmethane (DIM) is a beneficial dietary constituent that prevents breast tumors in rodents and is associated with decreased breast cancer risk in humans. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a toxic AhR ligand that is implicated in birth defects, infertility, and cancer. We analyzed MCF-7 cells to gain insight into how two AhR ligands can exert such fundamentally different health effects. We find that DIM and TCDD have differing abilities to activate the distinct AhR-controlled pathways. TCDD strongly induces AhR-dependent CYP1 gene expression, whereas DIM is a relatively weak CYP1 inducer. DIM strongly inhibits estrogen receptor-alpha expression and estrogen signaling, whereas TCDD has a notably weaker effect on these processes. Small interfering RNA knockdown of AhR confirms that the effects of DIM and TCDD are indeed AhR dependent. Our findings reveal that DIM and TCDD each elicit a unique pattern of change in pathways that control estrogen action; such patterns may determine if an AhR ligand has beneficial or adverse health effects.
Assuntos
Anticarcinógenos/uso terapêutico , Neoplasias da Mama/prevenção & controle , Receptores de Hidrocarboneto Arílico/metabolismo , Neoplasias da Mama/induzido quimicamente , Carcinógenos , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Dibenzodioxinas Policloradas , Ligação Proteica , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , RiscoRESUMO
Genistein (4',5,7-trihydroxyisoflavone) is the most abundant isoflavone found in the soybean. The effects of genistein on various cancer cell lines have been extensively studied but the precise molecular mechanisms are not known. We report here the epigenetic mechanism of the action of genistein on androgen-sensitive (LNCaP) and androgen-insensitive (DuPro) human prostate cancer cell lines. Genistein induced the expression of tumor suppressor genes p21 (WAF1/CIP1/KIP1) and p16 (INK4a) with a concomitant decrease in cyclins. There was a G(0)-G(1) cell cycle arrest in LNCaP cells and a G(2)-M arrest in DuPro cells after genistein treatment. Genistein also induced apoptosis in DuPro cells. DNA methylation analysis revealed the absence of p21 promoter methylation in both cell lines. The effect of genistein on chromatin remodeling has not been previously reported. We found that genistein increased acetylated histones 3, 4, and H3/K4 at the p21 and p16 transcription start sites. Furthermore, we found that genistein treatment also increased the expression of histone acetyl transferases that function in transcriptional activation. This is the first report on epigenetic regulation of various genes by genistein through chromatin remodeling in prostate cancer. Altogether, our data provide new insights into the epigenetic mechanism of the action of genistein that may contribute to the chemopreventive activity of this dietary isoflavone and have important implications for epigenetic therapy.
Assuntos
Cromatina/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Genisteína/farmacologia , Neoplasias da Próstata/genética , Acetilação , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatina/efeitos dos fármacos , DNA de Neoplasias/efeitos dos fármacos , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Perfilação da Expressão Gênica , Genes Supressores/efeitos dos fármacos , Genes Supressores de Tumor/efeitos dos fármacos , Histonas/efeitos dos fármacos , Histonas/metabolismo , Humanos , Masculino , Reação em Cadeia da Polimerase , Neoplasias da Próstata/patologiaRESUMO
Glutathione-S-transferases (GSTs) are metabolic enzymes that help detoxify and eliminate harmful chemicals. In prostate tumors, expression of GST pi (encoded by GSTP1) is frequently lost because of promoter hypermethylation. Here we analyze the native GSTP1 promoter in cancerous and noncancerous human prostate cells to identify structural features associated with its cancer-related transcriptional silencing. We find that in noncancerous prostate cells (RWPE-1 and PWR-1E) GSTP1 is constitutively expressed, not methylated, highly accessible, bound by transcription factors and associated with histones with activating modifications (histone H3 methylated at lysine 4 and acetylated histones H3 and H4). In contrast, in cancerous prostate cells (LNCaP) GSTP1 is not expressed, extensively methylated, inaccessible, lacks bound transcription factors and is not associated with histones with activating modifications. We do not detect significant levels of histones with repressive modifications (histone H3 methylated at lysine 9 or 27) on GSTP1 in any cell line indicating that they are not associated with cancer-related GSTP1 silencing. Treatment of LNCaP cells with 5-azacytidine restores activating histone modifications on GSTP1 and reactivates transcription. We conclude that, in the process of prostate carcinogenesis, activating histone modifications on GSTP1 are lost and the DNA becomes methylated and inaccessible resulting in transcriptional silencing.
Assuntos
Cromatina/ultraestrutura , Glutationa S-Transferase pi/genética , Regiões Promotoras Genéticas , Neoplasias da Próstata/genética , Sequência de Bases , Inativação Gênica , Humanos , Masculino , Dados de Sequência Molecular , Técnicas de Cultura de TecidosRESUMO
PURPOSE: We have previously reported that a C/A single nucleotide polymorphism at position -160 of the human E-cadherin gene promoter affects E-cadherin transcription. Although this single nucleotide polymorphism is associated with a number of human cancers, including prostate cancer, it is not known whether it has a role in race related prostate cancer. We hypothesized that allelic variation at this site may be associated with racial differences in the incidence and severity of prostate cancer. MATERIALS AND METHODS: We analyzed the genotypes of this single nucleotide polymorphism in a total of 135 prostate cancer samples from 86 white and 49 black American men, and in 237 samples from normal healthy controls, including 120 white and 117 black men, using the polymerase chain reaction-restriction fragment length polymorphism technique. RESULTS: Of normal controls black men had a higher frequency of the A allele and CA genotype than white men (26.5 and 39.3 vs 22.5 and 28.3, respectively). In white men A allele frequency was significantly higher in those with prostate cancer than in controls (p <0.05). White men carrying the A allele and AA genotype were at 1.99-fold (95% CI 1.29 to 3.08) and 3.04-fold (95% CI 1.26 to 7.32) higher risk for prostate cancer than carriers of the C allele and CC genotype. However, in black men the A allele was more frequent in controls than in patients with cancer and it was associated with a 2.4-fold decrease in prostate cancer risk (95% CI 0.22 to 0.81) compared to the C allele. CONCLUSIONS: The A allele of the E-cadherin -160 single nucleotide polymorphism represents a prostate cancer risk factor in white but not in black men.