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1.
Proc Natl Acad Sci U S A ; 120(31): e2301536120, 2023 08.
Article in English | MEDLINE | ID: mdl-37487069

ABSTRACT

Colorectal cancers (CRCs) form a heterogenous group classified into epigenetic and transcriptional subtypes. The basis for the epigenetic subtypes, exemplified by varying degrees of promoter DNA hypermethylation, and its relation to the transcriptional subtypes is not well understood. We link cancer-specific transcription factor (TF) expression alterations to methylation alterations near TF-binding sites at promoter and enhancer regions in CRCs and their premalignant precursor lesions to provide mechanistic insights into the origins and evolution of the CRC molecular subtypes. A gradient of TF expression changes forms a basis for the subtypes of abnormal DNA methylation, termed CpG-island promoter DNA methylation phenotypes (CIMPs), in CRCs and other cancers. CIMP is tightly correlated with cancer-specific hypermethylation at enhancers, which we term CpG-enhancer methylation phenotype (CEMP). Coordinated promoter and enhancer methylation appears to be driven by downregulation of TFs with common binding sites at the hypermethylated enhancers and promoters. The altered expression of TFs related to hypermethylator subtypes occurs early during CRC development, detectable in premalignant adenomas. TF-based profiling further identifies patients with worse overall survival. Importantly, altered expression of these TFs discriminates the transcriptome-based consensus molecular subtypes (CMS), thus providing a common basis for CIMP and CMS subtypes.


Subject(s)
Colorectal Neoplasms , Precancerous Conditions , Humans , Transcription Factors , Gene Expression Regulation , DNA Methylation , Epigenesis, Genetic
2.
J Med Chem ; 64(15): 11570-11596, 2021 08 12.
Article in English | MEDLINE | ID: mdl-34279934

ABSTRACT

Selective cyclooxygenase (COX)-2 inhibitors have been extensively studied for colorectal cancer (CRC) chemoprevention. Celecoxib has been reported to reduce the incidence of colorectal adenomas and CRC but is also associated with an increased risk of cardiovascular events. Here, we report a series of gut-restricted, selective COX-2 inhibitors characterized by high colonic exposure and minimized systemic exposure. By establishing acute ex vivo 18F-FDG uptake attenuation as an efficacy proxy, we identified a subset of analogues that demonstrated statistically significant in vivo dose-dependent inhibition of adenoma progression and survival extension in an APCmin/+ mouse model. However, in vitro-in vivo correlation analysis showed their chemoprotective effects were driven by residual systemic COX-2 inhibition, rationalizing their less than expected efficacies and highlighting the challenges associated with COX-2-mediated CRC disease chemoprevention.


Subject(s)
Antineoplastic Agents/pharmacology , Celecoxib/pharmacology , Colorectal Neoplasms/drug therapy , Cyclooxygenase 2 Inhibitors/pharmacology , Cyclooxygenase 2/metabolism , Etoricoxib/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Celecoxib/chemistry , Celecoxib/metabolism , Cell Proliferation/drug effects , Cell Survival/drug effects , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Cyclooxygenase 2 Inhibitors/chemistry , Cyclooxygenase 2 Inhibitors/metabolism , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Etoricoxib/chemistry , Etoricoxib/metabolism , Humans , Male , Mice , Mice, Inbred C57BL , Molecular Structure , Structure-Activity Relationship
3.
J Immunol ; 206(2): 422-431, 2021 01 15.
Article in English | MEDLINE | ID: mdl-33288543

ABSTRACT

Fibroblast-like synoviocytes (FLS), one of the main cell types of the rheumatoid arthritis (RA) synovium, possess phenotypic and molecular characteristics of transformed cells. JQ1, an inhibitor of the bromodomain and extra terminal domain family that includes BRD2, BRD3, BRD4, and BRDt, has shown efficacy in models of arthritis. We demonstrate that the active isomer of JQ1 but not its inactive isomer inhibits IL-1ß-induced RA-FLS activation and proliferation. To understand the mechanism of JQ1 action, we subjected JQ1-treated RA-FLS to transcriptional profiling and determined BRD2 and BRD4 cistromes by identifying their global chromatin binding sites. In addition, assay for transposable accessible chromatin by high throughput sequencing was employed to identify open and closed regions of chromatin in JQ1-treated RA-FLS. Through an integrated analysis of expression profiling, Brd2/Brd4 cistrome data, and changes in chromatin accessibility, we found that JQ1 inhibited key BRD2/BRD4 superenhancer genes, downregulated multiple crucial inflammatory pathways, and altered the genome-wide occupancy of critical transcription factors involved in inflammatory signaling. Our results suggest a pleiotropic effect of JQ1 on pathways that have shown to be individually efficacious in RA (in vitro, in vivo, and/or in humans) and provide a strong rationale for targeting BRD2/BRD4 for disease treatment and interception.

4.
Cancer Res ; 79(13): 3445-3454, 2019 07 01.
Article in English | MEDLINE | ID: mdl-31088836

ABSTRACT

Although ovarian cancer has a low incidence rate, it remains the most deadly gynecologic malignancy. Previous work has demonstrated that the DNMTi 5-Azacytidine (5AZA-C) activates type I interferon signaling to increase IFNγ+ T cells and natural killer (NK) cells and reduce the percentage of macrophages in the tumor microenvironment. To improve the efficacy of epigenetic therapy, we hypothesized that the addition of α-difluoromethylornithine (DFMO), an ornithine decarboxylase inhibitor, may further decrease immunosuppressive cell populations improving outcome. We tested this hypothesis in an immunocompetent mouse model for ovarian cancer and found that in vivo, 5AZA-C and DFMO, either alone or in combination, significantly increased survival, decreased tumor burden, and caused recruitment of activated (IFNγ+) CD4+ T cells, CD8+ T cells, and NK cells. The combination therapy had a striking increase in survival when compared with single-agent treatment, despite a smaller difference in recruited lymphocytes. Instead, combination therapy led to a significant decrease in immunosuppressive cells such as M2 polarized macrophages and an increase in tumor-killing M1 macrophages. In this model, depletion of macrophages with a CSF1R-blocking antibody reduced the efficacy of 5AZA-C + DFMO treatment and resulted in fewer M1 macrophages in the tumor microenvironment. These observations suggest our novel combination therapy modifies macrophage polarization in the tumor microenvironment, recruiting M1 macrophages and prolonging survival. SIGNIFICANCE: Combined epigenetic and polyamine-reducing therapy stimulates M1 macrophage polarization in the tumor microenvironment of an ovarian cancer mouse model, resulting in decreased tumor burden and prolonged survival.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Cystadenocarcinoma, Serous/immunology , Disease Models, Animal , Immunity, Innate/immunology , Macrophages/immunology , Ovarian Neoplasms/immunology , Tumor Microenvironment/immunology , Animals , Azacitidine/administration & dosage , Cystadenocarcinoma, Serous/drug therapy , Cystadenocarcinoma, Serous/metabolism , Cystadenocarcinoma, Serous/pathology , Eflornithine/administration & dosage , Female , Immunity, Innate/drug effects , Macrophages/drug effects , Mice , Mice, Inbred C57BL , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Polyamines/metabolism , Tumor Cells, Cultured , Tumor Microenvironment/drug effects
6.
PLoS One ; 13(8): e0200241, 2018.
Article in English | MEDLINE | ID: mdl-30138347

ABSTRACT

A model of B cell affinity selection is proposed, and an explanation of peripheral tolerance mechanisms through antibody repertoire editing is presented. We show that affinity discrimination between B cells is driven by a competition between obtaining T cell help and removal of B cells from the light zone, either through apoptosis or by a return to the dark zone of germinal centers. We demonstrate that this mechanism also allows for the negative selection of self reactive B cells and maintenance of B cell tolerance during the Germinal Center reaction. Finally, we demonstrate that clonal expansion upon return to the Germinal Center dark zone amplifies differences in the antigen affinity of B cells that survive the light zone.


Subject(s)
Antibody Affinity , B-Lymphocytes/immunology , Germinal Center/immunology , Models, Immunological , T-Lymphocytes/immunology , Animals , Antigens/metabolism , Apoptosis , Computer Simulation , Epitopes, B-Lymphocyte/metabolism , Epitopes, T-Lymphocyte/metabolism , Immune Tolerance
7.
Nat Commun ; 9(1): 1921, 2018 05 15.
Article in English | MEDLINE | ID: mdl-29765031

ABSTRACT

Epigenetics contributes to the pathogenesis of immune-mediated diseases like rheumatoid arthritis (RA). Here we show the first comprehensive epigenomic characterization of RA fibroblast-like synoviocytes (FLS), including histone modifications (H3K27ac, H3K4me1, H3K4me3, H3K36me3, H3K27me3, and H3K9me3), open chromatin, RNA expression and whole-genome DNA methylation. To address complex multidimensional relationship and reveal epigenetic regulation of RA, we perform integrative analyses using a novel unbiased method to identify genomic regions with similar profiles. Epigenomically similar regions exist in RA cells and are associated with active enhancers and promoters and specific transcription factor binding motifs. Differentially marked genes are enriched for immunological and unexpected pathways, with "Huntington's Disease Signaling" identified as particularly prominent. We validate the relevance of this pathway to RA by showing that Huntingtin-interacting protein-1 regulates FLS invasion into matrix. This work establishes a high-resolution epigenomic landscape of RA and demonstrates the potential for integrative analyses to identify unanticipated therapeutic targets.


Subject(s)
Arthritis, Rheumatoid/genetics , Epigenesis, Genetic , Fibroblasts/metabolism , Synoviocytes/metabolism , Adult , Aged , Arthritis, Rheumatoid/metabolism , Chromatin/genetics , Chromatin/metabolism , DNA Methylation , Female , Histone Code , Histones/genetics , Histones/metabolism , Humans , Male , Methylation , Middle Aged , Promoter Regions, Genetic
8.
Proc Natl Acad Sci U S A ; 114(51): E10981-E10990, 2017 12 19.
Article in English | MEDLINE | ID: mdl-29203668

ABSTRACT

Ovarian cancer is the most lethal of all gynecological cancers, and there is an urgent unmet need to develop new therapies. Epithelial ovarian cancer (EOC) is characterized by an immune suppressive microenvironment, and response of ovarian cancers to immune therapies has thus far been disappointing. We now find, in a mouse model of EOC, that clinically relevant doses of DNA methyltransferase and histone deacetylase inhibitors (DNMTi and HDACi, respectively) reduce the immune suppressive microenvironment through type I IFN signaling and improve response to immune checkpoint therapy. These data indicate that the type I IFN response is required for effective in vivo antitumorigenic actions of the DNMTi 5-azacytidine (AZA). Through type I IFN signaling, AZA increases the numbers of CD45+ immune cells and the percentage of active CD8+ T and natural killer (NK) cells in the tumor microenvironment, while reducing tumor burden and extending survival. AZA also increases viral defense gene expression in both tumor and immune cells, and reduces the percentage of macrophages and myeloid-derived suppressor cells in the tumor microenvironment. The addition of an HDACi to AZA enhances the modulation of the immune microenvironment, specifically increasing T and NK cell activation and reducing macrophages over AZA treatment alone, while further increasing the survival of the mice. Finally, a triple combination of DNMTi/HDACi plus the immune checkpoint inhibitor α-PD-1 provides the best antitumor effect and longest overall survival, and may be an attractive candidate for future clinical trials in ovarian cancer.


Subject(s)
Epigenesis, Genetic/drug effects , Immunomodulation/drug effects , Interferon Type I/metabolism , Ovarian Neoplasms/etiology , Ovarian Neoplasms/metabolism , Signal Transduction/drug effects , Animals , Antineoplastic Agents, Immunological , Azacitidine/pharmacology , Cell Line, Tumor , Disease Models, Animal , Female , Histone Deacetylase Inhibitors/pharmacology , Mice , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/pathology , Tumor Burden/drug effects , Tumor Burden/immunology , Xenograft Model Antitumor Assays
9.
J Immunol ; 199(12): 4091-4102, 2017 12 15.
Article in English | MEDLINE | ID: mdl-29127145

ABSTRACT

T cell expression of TIM-3 following Ag encounter has been associated with a continuum of functional states ranging from effector memory T cells to exhaustion. We have designed an in vitro culture system to specifically address the impact of anti-TIM-3/TIM-3 engagement on human Ag-specific CD8 T cells during a normal response to Ag and found that anti-TIM-3 treatment enhances T cell function. In our in vitro T cell culture system, MART1-specific CD8 T cells were expanded from healthy donors using artificial APCs. To ensure that the T cells were the only source of TIM-3, cells were rechallenged with peptide-loaded artificial APCs in the presence of anti-TIM-3 Ab. In these conditions, anti-TIM-3 treatment promotes generation of effector T cells as shown by acquisition of an activated phenotype, increased cytokine production, enhanced proliferation, and a transcription program associated with T cell differentiation. Activation of mTORC1 has been previously demonstrated to enhance CD8 T cell effector function and differentiation. Anti-TIM-3 drives CD8 T cell differentiation through activation of the mTORC1 as evidenced by increased levels of phosphorylated S6 protein and rhebl1 transcript. Altogether these findings suggest that anti-TIM-3, together with Ag, drives differentiation in favor of effector T cells via the activation of mTOR pathway. To our knowledge, this is the first report demonstrating that TIM-3 engagement during Ag stimulation directly influences T cell differentiation through mTORC1.


Subject(s)
Antigens/immunology , CD8-Positive T-Lymphocytes/immunology , Hepatitis A Virus Cellular Receptor 2/immunology , Immunologic Memory/immunology , Mechanistic Target of Rapamycin Complex 1/immunology , Antibodies, Monoclonal/pharmacology , Cell Division , Cells, Cultured , Gene Expression Profiling , Gene Expression Regulation/immunology , Humans , Lymphocyte Activation , Lymphokines/biosynthesis , Lymphokines/genetics , MART-1 Antigen/immunology , Phosphorylation , Protein Processing, Post-Translational , T-Cell Antigen Receptor Specificity , ras Proteins/biosynthesis , ras Proteins/genetics
10.
Oncol Rep ; 30(2): 707-14, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23708506

ABSTRACT

Lapatinib is a dual tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR) and human EGFR-2 (HER2) tyrosine kinase domains. To explore the potential utility of lapatinib for the treatment of esophageal squamous cell carcinoma (ESCC), we examined the expression profiles of EGFR and HER2 in tumor tissues and in paired adjacent non-neoplastic tissues from patients with ESCC. We evaluated the antitumor effects of lapatinib alone or in combination with oxaliplatin or 5-fluorouracil (5-FU) on a panel of primary ESCC cells in vitro with various levels of EGFR and HER2 expression. The in vivo effect of lapatinib alone or in combination with oxaliplatin or 5-FU was evaluated using a primary ESCC xenograft model. EGFR was overexpressed in 80.9% (76/94) of the ESCC samples, while 24.5% (23/94) of the samples overexpressed HER2. EGFR and HER2 co-overexpression was detected in 22.3% of samples (21/94). In vitro, the primary ESCC cells were more sensitive to lapatinib combined with 5-FU or oxaliplatin than to lapatinib alone. Lapatinib in combination with 5-FU had more potent antitumor effects in the primary ESCC xenograft model, and markedly reduced the phosphorylation of EGFR and HER2, compared with lapatinib alone or in combination with oxaliplatin. These data indicate that lapatinib has activity in EGFR- and/or HER2-expressing ESCC primary cells, and that lapatinib in combination with 5-FU may be a promising treatment strategy for patients with ESCC.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Carcinoma, Squamous Cell/drug therapy , Cell Proliferation/drug effects , Esophageal Neoplasms/drug therapy , Fluorouracil/pharmacology , Quinazolines/pharmacology , Animals , Antineoplastic Agents/administration & dosage , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Drug Synergism , ErbB Receptors/genetics , Esophageal Neoplasms/genetics , Esophageal Neoplasms/pathology , Esophageal Squamous Cell Carcinoma , Fluorouracil/administration & dosage , Humans , Lapatinib , Male , Mice , Mice, Nude , Organoplatinum Compounds/administration & dosage , Oxaliplatin , Phosphorylation/drug effects , Quinazolines/administration & dosage , Random Allocation , Receptor, ErbB-2/genetics , Transcriptome/drug effects , Xenograft Model Antitumor Assays/methods
11.
Exp Ther Med ; 5(1): 57-64, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23251242

ABSTRACT

Phosphatase and tensin homolog (PTEN) is a tumor suppressor involved in multiple cell processes. To investigate the role of PTEN in the development of gastric carcinoma, we determined the expression pattern of PTEN in primary gastric carcinoma and in paired adjacent non-neoplastic tissue. We also determined the correlation of PTEN expression with clinicopathological characteristics and patient survival. Overall, 159 gastric carcinomas and 151 paired adjacent non-neoplastic tissues were used in the present study. PTEN expression was determined using tissue microarrays and immunohistochemistry. The clinical sensitivity and specificity of PTEN expression were calculated using receiver operator characteristic curves. Results showed that the loss of cytoplasmic PTEN was significantly more frequent in carcinoma tissue compared with adjacent non-neoplastic tissue (62 vs. 5%, respectively; P<0.0001). PTEN expression was markedly downregulated in carcinoma tissues compared with adjacent non-neoplastic tissues. The loss of cytoplasmic PTEN expression was positively correlated with histological stage (P=0.016). The loss of nuclear or total PTEN, and downregulation of total PTEN expression, was significantly different between American Joint Committee on Cancer tumors of stage I and stages II-IV. A low cytoplasmic or total PTEN expression showed high clinical sensitivity and specificity for gastric carcinoma. However, PTEN expression was not significantly associated with overall or 3-year survival rates. The findings of the present study indicated that PTEN expression may be a molecular diagnostic marker for gastric cancer. Thus, the loss or reduced expression of PTEN potentially correlate with advanced stages of gastric carcinoma.

12.
Int J Mol Sci ; 13(8): 9980-9991, 2012.
Article in English | MEDLINE | ID: mdl-22949843

ABSTRACT

We aimed to investigate the expression pattern of phosphatase and tensin homolog (PTEN), to evaluate the relationship between PTEN expression and clinicopathological characteristics, including fatty acid synthase (FAS) expression, and to determine the correlations of PTEN and FAS expression with survival in Chinese patients with hepatocellular carcinoma (HCC). The expression patterns of PTEN and FAS were determined using tissue microarrays and immunohistochemistry. The expression of PTEN was compared with the clinicopathological characteristics of HCC, including FAS expression. Receiver operator characteristic curves were used to calculate the clinical sensitivity and specificity of PTEN expression. Kaplan-Meier survival curves were constructed to evaluate the correlations of PTEN loss and FAS overexpression with overall survival. We found that the loss of PTEN expression occurred predominantly in the cytoplasm, while FAS was mainly localized to the cytoplasm. Cytoplasmic and total PTEN expression levels were significantly decreased in HCC compared with adjacent non-neoplastic tissue (both, p < 0.0001). Decreased cytoplasmic and total PTEN expression showed significant clinical sensitivity and specificity for HCC (both, p < 0.0001). Downregulation of PTEN in HCC relative to non-neoplastic tissue was significantly correlated with histological grade (p = 0.043 for histological grades I-II versus grade III). Loss of total PTEN was significantly correlated with FAS overexpression (p = 0.014). Loss of PTEN was also associated with poor prognosis of patients with poorly differentiated HCC (p = 0.049). Moreover, loss of PTEN combined with FAS overexpression was associated with significantly worse prognosis compared with other HCC cases (p = 0.011). Our data indicate that PTEN may serve as a potential diagnostic and prognostic marker of HCC. Upregulating PTEN expression and inhibiting FAS expression may offer a novel therapeutic approach for HCC.


Subject(s)
Biomarkers, Tumor/metabolism , Carcinoma, Hepatocellular/metabolism , Fatty Acid Synthase, Type I/metabolism , Liver Neoplasms/metabolism , PTEN Phosphohydrolase/metabolism , Adult , Aged , Carcinoma, Hepatocellular/mortality , Carcinoma, Hepatocellular/secondary , China , Female , Gene Expression Regulation, Neoplastic , Humans , Immunoenzyme Techniques , Liver Neoplasms/mortality , Liver Neoplasms/pathology , Lymphatic Metastasis , Male , Middle Aged , Neoplasm Grading , Neoplasm Invasiveness , Neoplasm Staging , Prognosis , Survival Rate
13.
Cancer Biol Ther ; 13(12): 1185-94, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22892846

ABSTRACT

One of the hallmarks of cancer is metabolic deregulation. Many tumors display increased glucose uptake and breakdown through the process of aerobic glycolysis, also known as the Warburg effect. Less studied in cancer development and progression is the importance of the glutamine (Gln) pathway, which provides cells with a variety of essential products to sustain cell proliferation, such as ATP and macromolecules for biosynthesis. To this end Gln dependency was assessed in a panel of non-small cell lung cancer lines (NSCLC). Gln was found to be essential for the growth of cells with high rates of glutaminolysis, and after exploring multiple genes in the Gln pathway, GLS1 was found to be the key enzyme associated with this dependence. This dependence was confirmed by observing the rescue of decreased growth by exogenous addition of downstream metabolites of glutaminolysis. Expression of the GLS1 splice variant KGA was found to be decreased in tumors compared with normal lung tissue. Transient knock down of GLS1 splice variants indicated that loss of GAC had the most detrimental effect on cancer cell growth. In conclusion, NSCLC cell lines depend on Gln for glutaminolysis to a varying degree, in which the GLS1 splice variant GAC plays an essential role and is a potential target for cancer metabolism-directed therapy.


Subject(s)
Carcinoma, Non-Small-Cell Lung , Glutaminase , Glutamine/metabolism , Protein Isoforms , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Line, Tumor , Cell Proliferation , Glutaminase/genetics , Glutaminase/metabolism , Glycolysis , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Molecular Targeted Therapy , Protein Isoforms/genetics , Protein Isoforms/metabolism
14.
Clin Cancer Res ; 18(12): 3462-9, 2012 Jun 15.
Article in English | MEDLINE | ID: mdl-22421194

ABSTRACT

PURPOSE: We sought to evaluate the feasibility of detecting PIK3CA mutations in circulating tumor DNA (ctDNA) from plasma of patients with metastatic breast cancer using a novel technique called BEAMing. EXPERIMENTAL DESIGN: In a retrospective analysis, 49 tumor and temporally matched plasma samples from patients with breast cancer were screened for PIK3CA mutations by BEAMing. We then prospectively screened the ctDNA of 60 patients with metastatic breast cancer for PIK3CA mutations by BEAMing and compared the findings with results obtained by screening corresponding archival tumor tissue DNA using both sequencing and BEAMing. RESULTS: The overall frequency of PIK3CA mutations by BEAMing was similar in both patient cohorts (29% and 28.3%, respectively). In the retrospective cohort, the concordance of PIK3CA mutation status by BEAMing between formalin-fixed, paraffin-embedded (FFPE) samples and ctDNA from temporally matched plasma was 100% (34 of 34). In the prospective cohort, the concordance rate among 51 evaluable cases was 72.5% between BEAMing of ctDNA and sequencing of archival tumor tissue DNA. When the same archival tissue DNA was screened by both sequencing and BEAMing for PIK3CA mutations (n = 41 tissue samples), there was 100% concordance in the obtained results. CONCLUSIONS: Analysis of plasma-derived ctDNA for the detection of PIK3CA mutations in patients with metastatic breast cancer is feasible. Our results suggest that PIK3CA mutational status can change upon disease recurrence, emphasizing the importance of reassessing PIK3CA status on contemporary (not archival) biospecimens. These results have implications for the development of predictive biomarkers of response to targeted therapies.


Subject(s)
Breast Neoplasms/genetics , DNA, Neoplasm/blood , Phosphatidylinositol 3-Kinases/blood , Phosphatidylinositol 3-Kinases/genetics , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/genetics , Class I Phosphatidylinositol 3-Kinases , Cohort Studies , Female , Genetic Markers , Humans , Middle Aged , Mutation , Neoplasm Metastasis , Retrospective Studies
15.
Proc Natl Acad Sci U S A ; 109(8): 2724-9, 2012 Feb 21.
Article in English | MEDLINE | ID: mdl-22003129

ABSTRACT

Breast cancers are comprised of molecularly distinct subtypes that may respond differently to pathway-targeted therapies now under development. Collections of breast cancer cell lines mirror many of the molecular subtypes and pathways found in tumors, suggesting that treatment of cell lines with candidate therapeutic compounds can guide identification of associations between molecular subtypes, pathways, and drug response. In a test of 77 therapeutic compounds, nearly all drugs showed differential responses across these cell lines, and approximately one third showed subtype-, pathway-, and/or genomic aberration-specific responses. These observations suggest mechanisms of response and resistance and may inform efforts to develop molecular assays that predict clinical response.


Subject(s)
Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Breast Neoplasms/classification , Breast Neoplasms/drug therapy , Signal Transduction/drug effects , Breast Neoplasms/genetics , Cell Line, Tumor , Drug Screening Assays, Antitumor , Female , Gene Dosage/genetics , Humans , Models, Biological , Signal Transduction/genetics , Transcription, Genetic/drug effects
16.
Proc Natl Acad Sci U S A ; 108(43): 17773-8, 2011 Oct 25.
Article in English | MEDLINE | ID: mdl-21987798

ABSTRACT

Biallelic inactivation of cancer susceptibility gene BRCA1 leads to breast and ovarian carcinogenesis. Paradoxically, BRCA1 deficiency in mice results in early embryonic lethality, and similarly, lack of BRCA1 in human cells is thought to result in cellular lethality in view of BRCA1's essential function. To survive homozygous BRCA1 inactivation during tumorigenesis, precancerous cells must accumulate additional genetic alterations, such as p53 mutations, but this requirement for an extra genetic "hit" contradicts the two-hit theory for the accelerated carcinogenesis associated with familial cancer syndromes. Here, we show that heterozygous BRCA1 inactivation results in genomic instability in nontumorigenic human breast epithelial cells in vitro and in vivo. Using somatic cell gene targeting, we demonstrated that a heterozygous BRCA1 185delAG mutation confers impaired homology-mediated DNA repair and hypersensitivity to genotoxic stress. Heterozygous mutant BRCA1 cell clones also showed a higher degree of gene copy number loss and loss of heterozygosity in SNP array analyses. In BRCA1 heterozygous clones and nontumorigenic breast epithelial tissues from BRCA mutation carriers, FISH revealed elevated genomic instability when compared with their respective controls. Thus, BRCA1 haploinsufficiency may accelerate hereditary breast carcinogenesis by facilitating additional genetic alterations.


Subject(s)
Breast/cytology , Epithelial Cells/physiology , Genes, BRCA1 , Genomic Instability/genetics , Haploinsufficiency/genetics , Female , Gene Silencing , Genomic Instability/physiology , Heterozygote , Humans , In Situ Hybridization, Fluorescence , Polymorphism, Single Nucleotide , Sequence Deletion/genetics
17.
J Transl Med ; 9: 110, 2011 Jul 15.
Article in English | MEDLINE | ID: mdl-21762492

ABSTRACT

BACKGROUND: Aurora kinases play critical roles in mitosis and are being evaluated as therapeutic targets in cancer. GSK1070916 is a potent, selective, ATP competitive inhibitor of Aurora kinase B and C. Translation of predictive biomarkers to the clinic can benefit patients by identifying the tumors that are more likely to respond to therapies, especially novel inhibitors such as GSK1070916. METHODS: 59 Hematological cancer-derived cell lines were used as models for response where in vitro sensitivity to GSK1070916 was based on both time and degree of cell death. The response data was analyzed along with karyotype, transcriptomics and somatic mutation profiles to determine predictors of response. RESULTS: 20 cell lines were sensitive and 39 were resistant to treatment with GSK1070916. High chromosome number was more prevalent in resistant cell lines (p-value = 0.0098, Fisher Exact Test). Greater resistance was also found in cell lines harboring polyploid subpopulations (p-value = 0.00014, Unpaired t-test). A review of NOTCH1 mutations in T-ALL cell lines showed an association between NOTCH1 mutation status and chromosome number (p-value = 0.0066, Fisher Exact Test). CONCLUSIONS: High chromosome number associated with resistance to the inhibition of Aurora B and C suggests cells with a mechanism to bypass the high ploidy checkpoint are resistant to GSK1070916. High chromosome number, a hallmark trait of many late stage hematological malignancies, varies in prevalence among hematological malignancy subtypes. The high frequency and relative ease of measurement make high chromosome number a viable negative predictive marker for GSK1070916.


Subject(s)
Aza Compounds/pharmacology , Chromosomes, Human/genetics , Hematologic Neoplasms/enzymology , Hematologic Neoplasms/genetics , Indoles/pharmacology , Polyploidy , Protein Serine-Threonine Kinases/antagonists & inhibitors , Aurora Kinase B , Aurora Kinases , Cell Cycle/drug effects , Cell Death/drug effects , Cell Line, Tumor , Diploidy , Drug Resistance, Neoplasm/drug effects , Hematologic Neoplasms/pathology , Humans , Mutation/genetics , Phenotype , Prognosis , Protein Serine-Threonine Kinases/metabolism , Receptors, Notch/genetics
18.
Genomics ; 98(4): 296-301, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21600279

ABSTRACT

Identification of biomarkers for positive and negative predictors of response to cancer therapeutics can help direct clinical strategies. However, challenges with tissue availability and costs are significant limiting factors for diagnostic assays. To address these challenges, we have customized a high-throughput single nucleotide polymorphism genotyping assay with the objective of simultaneously surveying known somatic mutations and copy number alterations for translational studies in cancer. As constructed, this assay can interrogate 376 known somatic mutations and quantify copy number alterations of genes commonly implicated in tumorigenesis or progression. Validation of this assay on a panel of 321 cell lines demonstrates sensitivity to accurately detect mutations, robust accuracy in the presence of infiltrating normal tissue, and the ability to detect both DNA copy number amplifications and deletions. This technology, with its high sensitivity, small DNA requirements, and low costs is an attractive platform for biomarker exploration in cancer.


Subject(s)
Gene Dosage/genetics , High-Throughput Nucleotide Sequencing/methods , Neoplasms/genetics , Oncogenes/genetics , Point Mutation/genetics , Cell Line, Tumor , Genotype , Humans , Polymorphism, Single Nucleotide/genetics , Sensitivity and Specificity
19.
Mol Cancer Ther ; 9(7): 2079-89, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20571075

ABSTRACT

Polo-like kinases are a family of serine threonine kinases that are critical regulators of cell cycle progression and DNA damage response. Predictive biomarkers for the Plk1-selective inhibitor GSK461364A were identified by comparing the genomics and genetics of a panel of human cancer cell lines with their response to a drug washout followed by an outgrowth assay. In this assay, cell lines that have lost p53 expression or carry mutations in the TP53 gene tended to be more sensitive to GSK461364A. These more sensitive cell lines also had increased levels of chromosome instability, a characteristic associated with loss of p53 function. Further mechanistic studies showed that p53 wild-type (WT) and not mutant cells can activate a postmitotic tetraploidy checkpoint and arrest at pseudo-G(1) state after GSK461364A treatment. RNA silencing of WT p53 increased the antiproliferative activity of GSK461364A. Furthermore, silencing of p53 or p21/CDKN1A weakened the tetraploidy checkpoint in cells that survived mitotic arrest and mitotic slippage. As many cancer therapies tend to be more effective in p53 WT patients, the higher sensitivity of p53-deficient tumors toward GSK461364A could potentially offer an opportunity to treat tumors that are refractory to other chemotherapies as well as early line therapy for these genotypes.


Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Chromosomal Instability , Mutation , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Thiophenes/pharmacology , Tumor Suppressor Protein p53/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Humans , Immunoblotting , Inhibitory Concentration 50 , Mitosis/drug effects , Mitosis/genetics , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Polyploidy , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Tumor Suppressor Protein p53/metabolism , Polo-Like Kinase 1
20.
Curr Opin Genet Dev ; 20(3): 336-41, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20382522

ABSTRACT

DNA sequence and bioinformatics technology have enabled the analysis of the cancer genome, revealing the vast genetic complexity of this disease. The patterns of somatic mutations are a rich archaeological record of the insults received by the genome that have added to our understanding of the mutagenic process. However, very few frequently mutated genes have been identified with the majority of somatic mutational events occurring infrequently. These infrequent mutations, however, have been shown to effect well-defined biological pathways that are critical in driving the development and progression of human tumours, for example the MAPK and PI3K pathways. Current cancer sequencing studies are now providing somatic mutation data for distinct tumour types and subtypes, leading to the identification of disease-specific alterations and potential therapeutic targets.


Subject(s)
Genetic Predisposition to Disease/genetics , Genome, Human/genetics , Mutation , Neoplasms/genetics , Databases, Genetic , Humans , Sequence Analysis, DNA
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