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BACKGROUND: African-American (AA) men tend to present with more aggressive prostate cancer (Gleason score >7) than European-American (EA) men. Vitamin D and its metabolites are implicated in prostate cancer biology with vitamin D deficiency, indicated by its metabolite levels in serum or plasma, usually observed in AA men. OBJECTIVE: To determine if 1, 25-dihydroxy vitamin D3 [1,25(OH)2 D] plasma levels in AA and EA prostate cancer patients alter the risk of having aggressive prostate cancer. DESIGN: Research subjects from the North Carolina-Louisiana Prostate Cancer Project (AA n = 435 and EA n = 532) were included. Plasma metabolites 1,25(OH)2 D and 25-hydroxyvitamin D3 [25(OH)D] were measured using liquid chromatography with tandem mass spectrophotometry. Research subjects were classified into low (Gleason sum < 7, stage T1-T2, and Prostate-specific antigen (PSA) < 9 ng/mL) or high (Gleason sum > 8 or Gleason sum = 7 with 4 + 3, or PSA > 20 ng/mL, or Gleason sum = 7 and stage T3-T4) aggressive disease. RESULTS: Research subjects in the second and third tertiles of plasma levels of 1, 25(OH)2 D had lower odds of high aggressive prostate cancer (AA [ORT2vsT1 : 0.66, 95%CI: 0.39-1.12; ORT3vsT1 : 0.83, 95%CI: 0.49-1.41] and EA [ORT2vsT1 : 0.68, 95%CI: 0.41-1.11; ORT3vsT1 : 0.67, 95%CI: 0.40-1.11]) compared with the first tertile, though confidence intervals included the null. Greater 1,25(OH)2 D/25(OH)D molar ratios were associated with lower odds of high aggressive prostate cancer more evidently in AA (ORQ4vsQ1 : 0.45, CI: 0.24-0.82) than in EA (ORQ4vsQ1 : 0.64, CI: 0.35-1.17) research subjects. CONCLUSIONS: The 1,25(OH)2 D/25(OH)D molar ratio was associated with decreased risk of high aggressive prostate cancer in AA men, and possibly in EA men. Further studies analyzing vitamin D polymorphisms, vitamin D binding protein levels, and prostatic levels of these metabolites may be useful. These studies may provide a better understanding of the vitamin D pathway and its biological role underlying health disparities in prostate cancer.
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Calcitriol/sangue , Invasividade Neoplásica/patologia , Próstata/patologia , Neoplasias da Próstata/sangue , Vitamina D/análogos & derivados , Idoso , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Neoplasias da Próstata/patologia , Fatores de Risco , Vitamina D/sangueRESUMO
BACKGROUND: Class I histone deacetylases (HDACs) have been reported to be overexpressed in clear cell renal cell carcinoma (ccRCC), whereas the expression of class II HDACs is unknown. METHODS: Four isogenic cell lines C2/C2VHL and 786-O/786-OVHL with differential VHL expression are used in our studies. Cobalt chloride is used to mimic hypoxia in vitro. HIF-2α knockdowns in C2 and 786-O cells is used to evaluate the effect on HDAC 1 expression and activity. Invasion and migration assays are used to investigate the role of HDAC 1 and HDAC 6 expression in ccRCC cells. Comparisons are made between experimental groups using the paired T-test, the two-sample Student's T-test or one-way ANOVA, as appropriate. ccRCC and the TCGA dataset are used to observe the clinical correlation between HDAC 1 and HDAC 6 overexpression and overall and progression free survival. RESULTS: Our analysis of tumor and matched non-tumor tissues from radical nephrectomies showed overexpression of class I and II HDACs (HDAC6 only in a subset of patients). In vitro, both HDAC1 and HDAC6 over-expression increased cell invasion and motility, respectively, in ccRCC cells. HDAC1 regulated invasiveness by increasing matrix metalloproteinase (MMP) expression. Furthermore, hypoxia stimulation in VHL-reconstituted cell lines increased HIF isoforms and HDAC1 expression. Presence of hypoxia response elements in the HDAC1 promoter along with chromatin immunoprecipitation data suggests that HIF-2α is a transcriptional regulator of HDAC1 gene. Conversely, HDAC6 and estrogen receptor alpha (ERα) were co-localized in cytoplasm of ccRCC cells and HDAC6 enhanced cell motility by decreasing acetylated α-tubulin expression, and this biological effect was attenuated by either biochemical or pharmacological inhibition. Finally, analysis of human ccRCC specimens revealed positive correlation between HIF isoforms and HDAC. HDAC1 mRNA upregulation was associated with worse overall survival in the TCGA dataset. CONCLUSIONS: Taking together, these results suggest that HDAC1 and HDAC6 may play a role in ccRCC biology and could represent rational therapeutic targets.
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Carcinoma de Células Renais/patologia , Histona Desacetilase 1/metabolismo , Histona Desacetilases/metabolismo , Neoplasias Renais/patologia , Western Blotting , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/mortalidade , Linhagem Celular Tumoral , Movimento Celular , Imunoprecipitação da Cromatina , Intervalo Livre de Doença , Citometria de Fluxo , Imunofluorescência , Técnicas de Silenciamento de Genes , Desacetilase 6 de Histona , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Neoplasias Renais/metabolismo , Neoplasias Renais/mortalidade , Invasividade Neoplásica/patologia , Análise Serial de TecidosRESUMO
BACKGROUND: Prostate cancer is a significant health concern, particularly among African American (AA) men who exhibit higher incidence and mortality compared to European American (EA) men. Understanding the molecular mechanisms underlying these disparities is imperative for enhancing clinical management and achieving better outcomes. METHODS: Employing a multi-omics approach, we analyzed prostate cancer in both AA and EA men. Using Illumina methylation arrays and RNA sequencing, we investigated DNA methylation and gene expression in tumor and non-tumor prostate tissues. Additionally, Boolean analysis was utilized to unravel complex networks contributing to racial disparities in prostate cancer. RESULTS: When comparing tumor and adjacent non-tumor prostate tissues, we found that DNA hypermethylated regions are enriched for PRC2/H3K27me3 pathways and EZH2/SUZ12 cofactors. Olfactory/ribosomal pathways and distinct cofactors, including CTCF and KMT2A, were enriched in DNA hypomethylated regions in prostate tumors from AA men. We identified race-specific inverse associations of DNA methylation with expression of several androgen receptor (AR) associated genes, including the GATA family of transcription factors and TRIM63. This suggests that race-specific dysregulation of the AR signaling pathway exists in prostate cancer. To investigate the effect of AR inhibition on race-specific gene expression changes, we generated in-silico patient-specific prostate cancer Boolean networks. Our simulations revealed prolonged AR inhibition causes significant dysregulation of TGF-ß, IDH1, and cell cycle pathways specifically in AA prostate cancer. We further quantified global gene expression changes, which revealed differential expression of genes related to microtubules, immune function, and TMPRSS2-fusion pathways, specifically in prostate tumors of AA men. Enrichment of these pathways significantly correlated with an altered risk of disease progression in a race-specific manner. CONCLUSIONS: Our study reveals unique signaling networks underlying prostate cancer biology in AA and EA men, offering potential insights for clinical management strategies tailored to specific racial groups. Targeting AR and associated pathways could be particularly beneficial in addressing the disparities observed in prostate cancer outcomes in the context of AA and EA men. Further investigation into these identified pathways may lead to the development of personalized therapeutic approaches to improve outcomes for prostate cancer patients across different racial backgrounds.
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Neoplasias da Próstata , Receptores Androgênicos , Masculino , Humanos , Receptores Androgênicos/genética , Metilação de DNA , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Perfilação da Expressão Gênica , DNA/metabolismoRESUMO
Dihydrotestosterone (DHT) and testosterone (T), which mediate androgen receptor nuclear translocation and target gene transcription, are crucial androgens and essential molecular triggers required for the proliferation and survival of prostate cancer cells. Therefore, androgen metabolism is commonly targeted in the treatment of prostate cancer. Using a high-pressure liquid chromatographic assay with tandem mass spectral detection, we determined the serum levels of metabolites produced during DHT/T biosynthesis in African American (AA) and European American (EA) men with localized, therapy naïve prostate cancer. Serum progesterone and related metabolites were significantly lower in AA men than in EA men, and these differences were associated with rapid disease progression. Multivariate analysis revealed significant differences between a subset of intermediate androgen metabolites between AA and EA men and between men with <=3 + 4 and >=4 + 3 Gleason score disease. AA men have a significantly higher frequency of single nucleotide polymorphisms in CYP11B1 and CYP11B2, enzymes that regulate corticosterone-aldosterone conversion. Finally, higher levels of T and pregnenolone were associated with a lower risk of progression-free survival only in AA men. This work provides new insight into androgen metabolism and racial disparities in prostate cancer by presenting evidence of dysregulated androgen biosynthesis in therapy naïve disease that correlates with clinical variables.
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BACKGROUND: Currently, limited mouse models that mimic the clinical course of castrate resistant prostate development currently exist. Such mouse models are urgently required to conduct pre-clinical studies to assist in the understanding of disease progression and the development of rational therapeutic strategies to treat castrate resistant prostate cancer. METHODS: Wild type intact FVB male mice were injected by subcutaneous injection with Myc-CaP cells to establish androgen sensitive Myc-CaP tumors. Tumor bearing mice were castrated and resulting tumors serially passaged in pre-castrated FVB male mice to produce a bone fide Myc-CaP castrate resistant tumor. RESULTS: Immunohistochemical analysis revealed that initial androgen sensitive Myc-CaP tumors had strong nuclear transcriptional active androgen receptor expression, as indicated by marked c-MYC staining and were highly proliferative. Castration of tumor bearing animals resulted in cytoplasmic relocation of androgen receptor concurrent with loss of transcriptional activity and tumor proliferation. Serial passaging of castrate refractory Myc-CaP in pre-castrated male FVB mice resulted in the development of a bona fide castrate resistant Myc-CaP tumor which pheno-copied the original androgen sensitive parental Myc-CaP tumor. CONCLUSIONS: Developing a murine castrate transplant resistant tumor model that mimics the clinical course of human castrate resistant prostate cancer will create better opportunities to understand the development of castrate resistant prostate cancer and also allow for more rapid pre-clinical studies to stratify rational novel therapies for this lethal form of prostate cancer.
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Linhagem Celular Tumoral , Modelos Animais de Doenças , Camundongos , Neoplasias da Próstata/patologia , Animais , Masculino , Transplante de Neoplasias , Orquiectomia , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismoRESUMO
STAG2 (Stromal Antigen 2), in healthy somatic cells, functions in sister chromatid cohesion, DNA damage repair, and genome organization, but its role in muscle invasive bladder cancer (MIBC) remains unknown. Here, using whole-exome and targeted sequencing (n=119 bladder cancer clinical samples), we found several STAG2 mutations in MIBC that correlate with loss of protein expression. The analysis of a bladder cancer tissue microarray (n=346) revealed that decreased STAG2 protein expression is associated with improved overall and progression-free survival for MIBC patients. In mouse xenograft studies, STAG2 knockdown (KD) decelerated MIBC tumor growth, whereas STAG2 overexpression accelerated tumor growth. In cell line studies, STAG2 loss augmented treatment with cisplatin, a first-line therapy for MIBC. STAG2 KD or overexpression did not alter degree of aneuploidy, copy number variations, or cell cycle distribution. However, unbiased RNA sequencing analysis revealed that STAG2 KD altered gene expression. STAG2 KD led to significant downregulation of several gene sets, such as collagen containing extracellular matrix, external encapsulating structure organization, and regulation of chemotaxis. Therefore, we investigated the effect of STAG2 KD on cell migration and invasion in vitro. We found that STAG2 KD minimized cell speed, displacement, and invasion. Altogether, our results present a non-canonical function of STAG2 in promoting cell motility and invasion of MIBC cells. This work forms the basis for additional investigation into the role of STAG2 in transcriptional regulation and how it becomes dysregulated in STAG2-mutant MIBC.
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Variações do Número de Cópias de DNA , Neoplasias da Bexiga Urinária , Humanos , Animais , Camundongos , Proteínas de Ciclo Celular/genética , Antígenos Nucleares/genética , Neoplasias da Bexiga Urinária/genética , Segregação de Cromossomos , Fenótipo , Músculos/metabolismoRESUMO
The evolving paradigm of the molecular classification of bladder cancer requires models that represent the classifications with less heterogeneity. Robust transcriptome based molecular classifications are essential to address tumor heterogeneity. Patient derived models (PDMs) are a powerful preclinical tool to study specific tumor compartments. We tested if the consensus molecular subtype analysis was applicable to PDMs and evaluated the tumor compartment each model represents. PDMs derived from surgical specimens were established as xenografts (PDX), organoids (PDO), and spheroids (PDS). The surgical specimens and PDMs were molecularly characterized by RNA sequencing. PDMs that were established in immune deficient mice or in vitro significantly downregulated transcripts related to the immune and stromal compartments compared to the surgical specimens. However, PDMs upregulate a patient-specific bladder cancer cell signal which allowed for analysis of cancer cell pathways independent of the tumor microenvironment. Based on transcriptomic signatures, PDMs are more similar to their surgical specimen than the model type; indicating that the PDMs retained unique features of the tumor from which the PDM was derived. When comparing models, PDX models were the most similar to the surgical specimen, while PDO and PDS models were most similar to each other. When the consensus molecular subtype classification system was applied to both the surgical samples and the three PDMs, good concordance was found between all samples indicating that this system of classification can be applied to PDO and PDS models. PDMs reduce tumor heterogeneity and allow analysis of tumor cells while maintaining the gene expression profile representative of the original tumor.
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Receptor tyrosine kinase inhibitors have shown clinical benefit in clear cell renal cell carcinoma (ccRCC), but novel therapeutic strategies are needed. The angiopoietin/Tie2 and MET pathways have been implicated in tumor angiogenesis, metastases, and macrophage infiltration. In our study, we used trebananib, an angiopoietin 1/2 inhibitor, and a novel small-molecule MET kinase inhibitor in patient-derived xenograft (PDX) models of ccRCC. Our goal was to assess the ability of these compounds to alter the status of tumor-infiltrating macrophages, inhibit tumor growth and metastases, and prolong survival. Seven-week-old SCID mice were implanted subcutaneously or orthotopically with human ccRCC models. One month postimplantation, mice were treated with angiopoietin 1/2 inhibitor trebananib (AMG 386), MET kinase inhibitor, or combination. In our metastatic ccRCC PDX model, RP-R-02LM, trebananib alone, and in combination with a MET kinase inhibitor, significantly reduced lung metastases and M2 macrophage infiltration (P = 0.0075 and P = 0.0205, respectively). Survival studies revealed that treatment of the orthotopically implanted RP-R-02LM tumors yielded a significant increase in survival in both trebananib and combination groups. In addition, resection of the subcutaneously implanted primary tumor allowed for a significant survival advantage to the combination group compared with vehicle and both single-agent groups. Our results show that the combination of trebananib with a MET kinase inhibitor significantly inhibits the spread of metastases, reduces infiltrating M2-type macrophages, and prolongs survival in our highly metastatic ccRCC PDX model, suggesting a potential use for this combination therapy in treating patients with ccRCC.
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Angiopoietina-2/genética , Carcinoma de Células Renais/genética , Animais , Carcinoma de Células Renais/mortalidade , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Camundongos SCID , Metástase Neoplásica , Análise de Sobrevida , Microambiente TumoralRESUMO
Lysine-specific demethylase 6A (KDM6A) and members of the Switch/Sucrose Non-Fermentable (SWI/SNF) family are known to counteract the activity of Enhancer of Zeste Homolog 2 (EZH2), which is often overexpressed and is associated with poor prognosis in muscle-invasive bladder cancer. Here we provide evidence that alterations in chromatin modifying enzymes, including KDM6A and members of the SWI/SNF complex, are frequent in muscle-invasive bladder cancer. We exploit the loss of function mutations in KDM6A and SWI/SNF complex to make bladder cancer cells susceptible to EZH2-based epigenetic therapy that activates an immune response to drive tumor cell differentiation and death. We reveal a novel mechanism of action of EZH2 inhibition, alone and in combination with cisplatin, which induces immune signaling with the largest changes observed in interferon gamma (IFN-γ). This upregulation is a result of activated natural killer (NK) signaling as demonstrated by the increase in NK cell-associated genes MIP-1α, ICAM1, ICAM2, and CD86 in xenografts treated with EZH2 inhibitors. Conversely, EZH2 inhibition results in decreased expression of pluripotency markers, ALDH2 and CK5, and increased cell death. Our results reveal a novel sensitivity of muscle-invasive bladder cancer cells with KMD6A and SWI/SNF mutations to EZH2 inhibition alone and in combination with cisplatin. This sensitivity is mediated through increased NK cell-related signaling resulting in tumor cell differentiation and cell death.
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Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/imunologia , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Proteína Potenciadora do Homólogo 2 de Zeste/imunologia , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Nus , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Intrinsic and/or acquired resistance to cisplatin is a significant obstacle in the treatment of muscle-invasive bladder cancer. p73, a p53 homolog and determinant of chemosensitivity, is rarely mutated in bladder cancer (BC). However p73 expression and therefore function can be repressed through epigenetic changes. In this study, we sought to identify DNA methylation status of p73, expression of TAp73 isoform, and their role in cisplatin sensitivity in BC. Primary tumor samples from 338 bladder cancer patients showed decreased TAp73 expression in MIBC compared to superficial BC. Low TAp73 protein expression was associated with shorter overall survival. To investigate if the loss of expression was methylation dependent, we utilized Illumina 450K methylation arrays to interrogate over 150 BC patient samples. We found 12 distinct CpGs in the p73 gene locus that were hypermethylated in tumors compared to adjacent normal tissues. Patients with high p73 promoter methylation specifically at CpG site cg07382920 had worse survival. In vitro, treatment with a DNA demethylating agent, decitabine (DAC), decreased TAp73 methylation and upregulated expression in both CR-T24 (cisplatin resistant T24 cells) and wild type T24 cells. Furthermore, treatment with DAC increased cisplatin response in wild type T24 and CR-T24. Our studies indicate that TAp73 expression and P1 promoter methylation, specifically at the cg073892920 site, may have prognostic and diagnostic value in MIBC. In the setting of P1 promoter hypermethylation, DAC could be used as a potentiating agent of cisplatin-based chemotherapy.
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Biomarcadores Tumorais/genética , Metilação de DNA/efeitos dos fármacos , Proteína Tumoral p73/genética , Neoplasias da Bexiga Urinária/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Cisplatino/efeitos adversos , Proteínas de Ligação a DNA/genética , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos/genética , Epigenômica/métodos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética , Proteínas Supressoras de Tumor/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologiaRESUMO
Cancer cells set in motion transcriptomic programs allowing for adaptation and growth in immunocompromised mice to form xenografts, a frequently used tool in cancer research. 2D cultures may not be representative of tumors growing in a complex host microenvironment. This can result in different responses to the same agent tested in vitro and in vivo which impedes the process of developing novel therapeutics. Understanding the transition cells undergo from 2D cell culture to a 3D host microenvironment will help in developing and choosing appropriate models for pre-clinical studies. Our study characterized the transcriptome of a three frequently used muscle-invasive bladder cancer cell lines HT1376, T24 and UM-UC-3 grown in culture and xenografts in nude mice. We found that bladder cancer cells undergo few transcriptomic changes when transitioned from 2D cell culture to xenografts in nude mice. UM-UC-3 cells have the least transcriptomic alterations followed by T24 and HT1376 cells. Respective xenografts cluster with their parental cell lines rather than other xenografts or cell lines. We applied established bladder cancer molecular subtypes to our data and found that UM-UC-3, containing the least transcriptomic alterations, most closely resembled the basal-like molecular subtype of bladder cancer. HT1376 and T24 have mixed basal and luminal molecular signatures. Our studies suggest this subset of bladder cancer cell lines and derived xenografts maintain similar transcriptomic profiles in both 2D culture and 3D xenografts and can be used interchangeably in pre-clinical studies.
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Aberrant DNA methylation observed in cancer can provide survival benefits to cells by silencing genes essential for anti-tumor activity. DNA-demethylating agents such as Decitabine (DAC)/Azacitidine (AZA) activate otherwise silenced tumor suppressor genes, alter immune response and epigenetically reprogram tumor cells. In this study, we show that non-cytotoxic nanomolar DAC concentrations modify the bladder cancer transcriptome to activate NOTCH1 at the mRNA and protein level, increase double-stranded RNA sensors and CK5-dependent differentiation. Importantly, DAC treatment increases ICN1 expression (the active intracellular domain of NOTCH1) significantly inhibiting cell proliferation and causing changes in cell size inducing morphological alterations reminiscent of senescence. These changes were not associated with ß-galactosidase activity or increased p16 levels, but instead were associated with substantial IL-6 release. Increased IL-6 release was observed in both DAC-treated and ICN1 overexpressing cells as compared to control cells. Exogenous IL-6 expression was associated with a similar enlarged cell morphology that was rescued by the addition of a monoclonal antibody against IL-6. Treatment with DAC, overexpression with ICN1 or addition of exogenous IL-6 showed CK5 reduction, a surrogate marker of differentiation. Overall this study suggests that in MIBC cells, DNA hypomethylation increases NOTCH1 expression and IL-6 release to induce CK5-related differentiation.
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Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Regulação Neoplásica da Expressão Gênica , Interleucina-6/genética , Receptor Notch1/genética , Neoplasias da Bexiga Urinária/genética , Anticorpos Monoclonais/farmacologia , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/imunologia , Decitabina , Epigênese Genética , Humanos , Interleucina-6/antagonistas & inibidores , Interleucina-6/imunologia , Queratina-5/genética , Queratina-5/metabolismo , Músculo Liso/imunologia , Músculo Liso/patologia , Invasividade Neoplásica , Receptor Notch1/metabolismo , Transdução de Sinais , Resultado do Tratamento , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/patologia , beta-Galactosidase/genética , beta-Galactosidase/imunologiaRESUMO
PURPOSE: Effective systemic therapeutic options are limited for bladder cancer. In this preclinical study we tested whether bladder cancer gene alterations may be predictive of treatment response. EXPERIMENTAL DESIGN: We performed genomic profiling of two bladder cancer patient derived tumor xenografts (PDX). We optimized the exome sequence analysis method to overcome the mouse genome interference. RESULTS: We identified a number of somatic mutations, mostly shared by the primary tumors and PDX. In particular, BLCAb001, which is less responsive to cisplatin than BLCAb002, carried non-sense mutations in several genes associated with cisplatin resistance, including MLH1, BRCA2, and CASP8. Furthermore, RNA-Seq analysis revealed the overexpression of cisplatin resistance associated genes such as SLC7A11, TLE4, and IL1A in BLCAb001. Two different PIK3CA mutations, E542K and E545K, were identified in BLCAb001 and BLCAb002, respectively. Thus, we tested whether the genomic profiling was predictive of response to a dual PI3K/mTOR targeting agent, LY3023414. Despite harboring similar PIK3CA mutations, BLCAb001 and BLCAb002 exhibited differential response, both in vitro and in vivo. Sustained target modulation was observed in the sensitive model BLCAb002 but not in BLCAb001, as well as decreased autophagy. Interestingly, computational modelling of mutant structures and affinity binding to PI3K revealed that E542K mutation was associated with weaker drug binding than E545K. CONCLUSIONS: Our results suggest that the presence of activating PIK3CA mutations may not necessarily predict in vivo treatment response to PI3K targeted therapies, while specific gene alterations may be predictive for cisplatin response in bladder cancer models and, potentially, in patients as well.
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Antineoplásicos/farmacologia , Cisplatino/farmacologia , Perfilação da Expressão Gênica , Genômica , Inibidores de Fosfoinositídeo-3 Quinase , Neoplasias da Bexiga Urinária/genética , Animais , Linhagem Celular Tumoral , Análise Mutacional de DNA , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Genômica/métodos , Humanos , Imuno-Histoquímica , Camundongos , Mutação , Fenótipo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Reprodutibilidade dos Testes , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Sequenciamento Completo do Genoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Progression of aggressive prostate cancers (PCa) with androgen receptor splice variants or neuroendrocrine features is currently untreatable in the clinic. Therefore novel therapies are urgently required. We conducted RNA-seq using tumors from a unique murine transplant mouse model which spontaneously progresses to metastatic disease. Differential gene expression analysis revealed a significant increase of topoisomerase IIα, Top2a (Top2a) in metastatic tumors. Interrogation of human data revealed that increased Top2a expression in primary tumors selected patients with more aggressive disease. Further, significant positive correlation was observed between Top2a and the histone methyltransferase, Ezh2. Combination of the Top2 poison etoposide with the Ezh2 inhibitor GSK126 or DZNep significantly increased cell death in vitro in murine and human prostate cancer cell lines. Additionally, combination therapy extended time to progression and increased therapeutic efficacy in vivo. Overall, our studies demonstrate that patients screened for Top2a and Ezh2 expression would exhibit significant response to a combinational treatment involving low dose etoposide combined with Ezh2 inhibition. In addition, our data suggests that this combination therapeutic strategy is beneficial against aggressive PCa, and provides strong rationale for continued clinical development.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Biomarcadores Tumorais/antagonistas & inibidores , Proteínas de Ligação a DNA/antagonistas & inibidores , Epigênese Genética , Neoplasias da Próstata/tratamento farmacológico , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Intervalo Livre de Doença , Proteína Potenciadora do Homólogo 2 de Zeste , Etoposídeo/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Indóis/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Proteínas de Ligação a Poli-ADP-Ribose , Complexo Repressor Polycomb 2/antagonistas & inibidores , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Medicina de Precisão , Valor Preditivo dos Testes , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Piridonas/farmacologia , RNA Mensageiro/metabolismo , Fatores de Tempo , Inibidores da Topoisomerase II/farmacologia , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Alternative pathways to the VEGF, such as hepatocyte growth factor or HGF/c-met, are emerging as key players in tumor angiogenesis and resistance to anti-VEGF therapies. The aim of this study was to assess the effects of a combination strategy targeting the VEGF and c-met pathways in clear cell renal cell carcinoma (ccRCC) models. Male SCID mice (8/group) were implanted with 786-O tumor pieces and treated with either a selective VEGF receptor tyrosine kinase inhibitor, axitinib (36 mg/kg, 2×/day); a c-met inhibitor, crizotinib (25 mg/kg, 1×/day); or combination. We further tested this drug combination in a human ccRCC patient-derived xenograft, RP-R-01, in both VEGF-targeted therapy-sensitive and -resistant models. To evaluate the resistant phenotype, we established an RP-R-01 sunitinib-resistant model by continuous sunitinib treatment (60 mg/kg, 1×/day) of RP-R-01-bearing mice. Treatment with single-agent crizotinib reduced tumor vascularization but failed to inhibit tumor growth in either model, despite also a significant increase of c-met expression and phosphorylation in the sunitinib-resistant tumors. In contrast, axitinib treatment was effective in inhibiting angiogenesis and tumor growth in both models, with its antitumor effect significantly increased by the combined treatment with crizotinib, independently from c-met expression. Combination treatment also induced prolonged survival and significant tumor growth inhibition in the 786-O human RCC model. Overall, our results support the rationale for the clinical testing of combined VEGF and HGF/c-met pathway blockade in the treatment of ccRCC, both in first- and second-line setting.
Assuntos
Carcinoma de Células Renais/tratamento farmacológico , Imidazóis/administração & dosagem , Indazóis/administração & dosagem , Indóis/administração & dosagem , Neoplasias Renais/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Pirazóis/administração & dosagem , Piridinas/administração & dosagem , Pirróis/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Axitinibe , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Crizotinibe , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Neoplasias Renais/metabolismo , Masculino , Camundongos , Camundongos SCID , Proteínas Proto-Oncogênicas c-met/metabolismo , Sunitinibe , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A major barrier for cancer immunotherapy is the presence of suppressive cell populations in patients with cancer, such as myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM), which contribute to the immunosuppressive microenvironment that promotes tumor growth and metastasis. Tasquinimod is a novel antitumor agent that is currently at an advanced stage of clinical development for treatment of castration-resistant prostate cancer. A target of tasquinimod is the inflammatory protein S100A9, which has been demonstrated to affect the accumulation and function of tumor-suppressive myeloid cells. Here, we report that tasquinimod provided a significant enhancement to the antitumor effects of two different immunotherapeutics in mouse models of cancer: a tumor vaccine (SurVaxM) for prostate cancer and a tumor-targeted superantigen (TTS) for melanoma. In the combination strategies, tasquinimod inhibited distinct MDSC populations and TAMs of the M2-polarized phenotype (CD206(+)). CD11b(+) myeloid cells isolated from tumors of treated mice expressed lower levels of arginase-1 and higher levels of inducible nitric oxide synthase (iNOS), and were less immunosuppressive ex vivo, which translated into a significantly reduced tumor-promoting capacity in vivo when these cells were coinjected with tumor cells. Tumor-specific CD8(+) T cells were increased markedly in the circulation and in tumors. Furthermore, T-cell effector functions, including cell-mediated cytotoxicity and IFNγ production, were potentiated. Taken together, these data suggest that pharmacologic targeting of suppressive myeloid cells by tasquinimod induces therapeutic benefit and provide the rationale for clinical testing of tasquinimod in combination with cancer immunotherapies.
Assuntos
Antineoplásicos/uso terapêutico , Imunoterapia/métodos , Melanoma Experimental/terapia , Neoplasias da Próstata/terapia , Quinolinas/uso terapêutico , Animais , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/uso terapêutico , Castração , Terapia Combinada , Avaliação Pré-Clínica de Medicamentos/métodos , Tolerância Imunológica/efeitos dos fármacos , Masculino , Melanoma Experimental/imunologia , Camundongos Endogâmicos C57BL , Células Mieloides/imunologia , Transplante de Neoplasias , Neoplasias da Próstata/imunologia , Quinolonas , Subpopulações de Linfócitos T/imunologiaRESUMO
Sunitinib is considered a first-line therapeutic option for patients with advanced clear cell renal cell carcinoma (ccRCC). Despite sunitinib's clinical efficacy, patients eventually develop drug resistance and disease progression. Herein, we tested the hypothesis whether initial sunitinib resistance may be transient and could be overcome by dose increase. In selected patients initially treated with 50 mg sunitinib and presenting with minimal toxicities, sunitinib dose was escalated to 62.5 mg and/or 75 mg at the time of tumor progression. Mice bearing two different patient-derived ccRCC xenografts (PDX) were treated 5 days per week with a dose-escalation schema (40-60-80 mg/kg sunitinib). Tumor tissues were collected before dose increments for immunohistochemistry analyses and drug levels. Selected intrapatient sunitinib dose escalation was safe and several patients had added progression-free survival. In parallel, our preclinical results showed that PDXs, although initially responsive to sunitinib at 40 mg/kg, eventually developed resistance. When the dose was incrementally increased, again we observed tumor response to sunitinib. A resistant phenotype was associated with transient increase of tumor vasculature despite intratumor sunitinib accumulation at higher dose. In addition, we observed associated changes in the expression of the methyltransferase EZH2 and histone marks at the time of resistance. Furthermore, specific EZH2 inhibition resulted in increased in vitro antitumor effect of sunitinib. Overall, our results suggest that initial sunitinib-induced resistance may be overcome, in part, by increasing the dose, and highlight the potential role of epigenetic changes associated with sunitinib resistance that can represent new targets for therapeutic intervention.
Assuntos
Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/genética , Resistencia a Medicamentos Antineoplásicos , Epigênese Genética , Indóis/uso terapêutico , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , Pirróis/uso terapêutico , Animais , Carcinoma de Células Renais/irrigação sanguínea , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Proteína Potenciadora do Homólogo 2 de Zeste , Epigênese Genética/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Indóis/sangue , Indóis/farmacologia , Neoplasias Renais/irrigação sanguínea , Neoplasias Renais/patologia , Camundongos SCID , Microvasos/efeitos dos fármacos , Microvasos/patologia , Complexo Repressor Polycomb 2/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirróis/sangue , Pirróis/farmacologia , Sunitinibe , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
First line treatment of patients with castrate resistant prostate cancer (CRPC) primarily involves administration of docetaxel chemotherapy. Unfortunately, resistance to docetaxel therapy is an ultimate occurrence. Alterations in androgen receptor (AR) expression and signaling are associated mechanisms underlying resistance to docetaxel treatment in CRPC. Heat shock protein 90 (Hsp90) is a molecular chaperone, which regulates the activation, maturation and stability of critical signaling proteins involved in prostate cancer, including the AR. This knowledge and recent advances in compound design and development have highlighted Hsp90 as an attractive therapeutic target for the treatment of CRPC. We recently reported the development of a MYC-CaP castrate resistant (MYC-CaP/CR) transplant tumor model, which expresses amplified wild type AR. Within, we report that a second generation Hsp90 inhibitor, NVP-AUY922, inhibits cell growth and significantly induces cell death in MYC-CaP/CR and Pten-CaP/cE2 cell lines. NVP-AUY922 induced proteasome degradation of AR, though interestingly does not require loss of AR protein to inhibit AR transcriptional activity. Further, NVP-AUY922 increased docetaxel toxicity in MYC-CaP/CR and Pten-CaP/cE2 cell lines in vitro. Finally, NVP-AUY922/docetaxel combination therapy in mice bearing MYC-CaP/CR tumors resulted in greater anti-tumor activity compared to single treatment. This study demonstrates that NVP-AUY922 elicits potent activity towards AR signaling and augments chemotherapy response in a mouse model of CRPC, providing rationale for the continued clinical development of Hsp90 inhibitors in clinical trials for treatment of CRPC patients.
Assuntos
Antineoplásicos/uso terapêutico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Isoxazóis/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Resorcinóis/uso terapêutico , Taxoides/uso terapêutico , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Docetaxel , Resistencia a Medicamentos Antineoplásicos , Quimioterapia Combinada , Proteínas de Choque Térmico HSP90/metabolismo , Isoxazóis/farmacologia , Masculino , Camundongos , Camundongos SCID , Neoplasias de Próstata Resistentes à Castração/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Resorcinóis/farmacologia , Taxoides/farmacologia , Transcrição Gênica , Transplante HeterólogoRESUMO
Recent investigations of renal cell carcinoma (RCC) have revealed several epigenetic modifications, as well as alterations in the genes and enzymes that regulate these changes. Preclinical models have revealed that histone gene modifiers and epigenetic alterations may play a critical role in RCC tumorigenesis. Specific changes in DNA methylation and mutations of histone modifiers have been identified and may be associated with an aggressive phenotype. In addition, the potential of reversing the effects of these enzymes and hence reversing the cellular epigenetic landscape to a "normal phenotype" have led to an increasing interest in developing targeted chromatin remodeling agents. However, the translation of the understanding of these changes to the clinic for the treatment of RCC has posed significant challenges, partly due to tumor heterogeneity. This review describes the aberrant histone and DNA alterations recently reported in RCC and highlights the potential targeted chromatin remodeling therapies in the management of this disease.
Assuntos
Carcinogênese/genética , Carcinoma de Células Renais/tratamento farmacológico , Montagem e Desmontagem da Cromatina/genética , Metilação de DNA/genética , Inibidores de Histona Desacetilases/uso terapêutico , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Epigênese Genética/genética , Regulação Neoplásica da Expressão Gênica/genética , Histonas/genética , Humanos , Terapia de Alvo MolecularRESUMO
In 2012, an estimated 64,770 men and women were diagnosed with malignancy of the kidney and renal pelvis, of which 13,570 succumbed to their disease. Common genetic aberrations in renal cell carcinomas (RCCs) include loss of function of the VHL gene in clear-cell RCC, overexpression of the c-MET gene in papillary RCC type I, deficiency in the FH gene in papillary RCC type II and loss of heterozygozity of the BHD gene in chromophobe RCC. Recent studies illustrate epigenetic silencing of VHL, as well as alterations in histone modifications and their governing enzymes. The possibility of reversing these epigenetic marks has resulted in efforts to target these changes by utilizing inhibitors of HDACs, DNA methyltransferases and, recently, histone methyltransferases in preclinical and clinical studies. This article focuses on potential therapeutic interventions, and the implications of histone modifications and related enzyme alterations in RCC.