ABSTRACT
PURPOSE: Safety and efficacy of acapatamab, a prostate-specific membrane antigen (PSMA) x CD3 bispecific T-cell engager were evaluated in a first-in-human study in metastatic castration-resistant prostate cancer (mCRPC). PATIENTS AND METHODS: Patients with mCRPC refractory to androgen receptor pathway inhibitor therapy and taxane-based chemotherapy received target acapatamab doses ranging from 0.003 to 0.9 mg in dose exploration (seven dose levels) and 0.3 mg (recommended phase II dose) in dose expansion intravenously every 2 weeks. Safety (primary objective), pharmacokinetics, and antitumor activity (secondary objectives) were assessed. RESULTS: In all, 133 patients (dose exploration, n = 77; dose expansion, n = 56) received acapatamab. Cytokine release syndrome (CRS) was the most common treatment-emergent adverse event seen in 97.4% and 98.2% of patients in dose exploration and dose expansion, respectively; grade ≥ 3 was seen in 23.4% and 16.1%, respectively. Most CRS events were seen in treatment cycle 1; incidence and severity decreased at/beyond cycle 2. In dose expansion, confirmed prostate-specific antigen (PSA) responses (PSA50) were seen in 30.4% of patients and radiographic partial responses in 7.4% (Response Evaluation Criteria in Solid Tumors 1.1). Median PSA progression-free survival (PFS) was 3.3 months [95% confidence interval (CI): 3.0-4.9], radiographic PFS per Prostate Cancer Clinical Trials Working Group 3 was 3.7 months (95% CI: 2.0-5.4). Acapatamab induced T-cell activation and increased cytokine production several-fold within 24 hours of initiation. Treatment-emergent antidrug antibodies were detected in 55% and impacted serum exposures in 36% of patients in dose expansion. CONCLUSIONS: Acapatamab was safe and tolerated and had a manageable CRS profile. Preliminary signs of efficacy with limited durable antitumor activity were observed. Acapatamab demonstrated pharmacokinetic and pharmacodynamic activity.
Subject(s)
Antineoplastic Agents , Prostatic Neoplasms, Castration-Resistant , Male , Humans , Prostatic Neoplasms, Castration-Resistant/pathology , Prostate-Specific Antigen , Half-Life , Treatment Outcome , Antineoplastic Agents/therapeutic use , Androgen Receptor Antagonists/therapeutic use , T-Lymphocytes/metabolismABSTRACT
Introduction The elevation of aminotransferase levels is regarded as an indicator of hepatocellular injury. The objective of this study was to describe real-world incidence of elevated aminotransferase levels with or without bilirubin elevation among patients treated with immune checkpoint inhibitors (ICIs) for solid tumors. Methods This retrospective cohort study used an electronic health record database representing > 1.5 million active United States (US) cancer patients and included patients diagnosed with any cancer between January 1, 2014 and March 31, 2019, and treated with one or more ICIs such as ipilimumab, tremelimumab, nivolumab, pembrolizumab, atezolizumab, durvalumab, and avelumab. The frequency, onset, duration, management of grade ≥ 3 elevation of aminotransferase levels with or without bilirubin elevation events, progression rate from isolated elevation of aminotransferase levels (IAT) to elevated aminotransferase levels with elevated bilirubin (ATWB), and mortality were described. Results Overall, 69,140 patients received 85,433 treatment courses. A total of 1,799 (2.11%) IAT and 441 (0.52%) ATWB events were observed during treatment courses. The median onset was 51 and 42 days for IAT and ATWB, respectively, across treatment courses, and the median duration of both was approximately seven days. Approximately 5% (n=96) of IAT events progressed to ATWB in a median time of 11 days. The proportion of patients who received corticosteroids after elevated aminotransferase levels with or without bilirubin was ~37% (n=671/1,799 of IAT and n=147/441 of ATWB) and ~8% discontinued ICI treatment (n=118/1,799 of IAT and n=43/441 of ATWB). About 46% (n=68/147) of ATWB and and 25% (n=172/671) of IAT events treated with steroids led to death within 45 days. Similarly, 49% (n=21/43) of ATWB and 35% (n=42/118) of IAT events leading to treatment discontinuation led to death within 45 days. Conclusions Real-world data from oncology clinics in US suggest low incidence of grade ≥ 3 elevated aminotransferase levels with or without bilirubin elevation following treatment with ICIs. In most cases, ICI treatment was not discontinued and management of elevated aminotransferases consisted of corticosteroid treatment in one-third of cases.
ABSTRACT
Antibody-drug conjugates (ADCs) are a new class of therapeutics that use antibodies to deliver potent cytotoxic drugs selectively to cancer cells. CD203c, an ecto-nucleotide pyrophosphatase-phosphodiesterase 3, is overexpressed on neoplastic mast cells (MCs) in systemic mastocytosis (SM), thus representing a promising target for antibody-mediated therapy. In this study, we have found that human neoplastic MC lines (ROSAKIT D816V and ROSAKIT D816V-Gluc), which express high levels of CD203c, are highly and specifically sensitive to the antiproliferative effects of an ADC against CD203c (AGS-16C3F). In these cell lines, AGS-16C3F induced cell apoptosis at very low concentrations. To characterize the effects of AGS-16C3F on leukemia progression in vivo, ROSAKIT D816V-Gluc NOD-SCID γ mouse models of advanced SM (AdvSM) were treated with AGS-16C3F or an ADC control for 2 weeks. Whereas AGS-16C3F had no apparent toxicity in xenotransplanted mice, in vivo neoplastic MC burden significantly decreased in both hematopoietic and nonhematopoietic organs. Furthermore, animals treated with AGS-16C3F had prolonged survival compared with the animals treated with control ADC, and AGS-16C3F efficiently prevented disease relapse. In conclusion, these preclinical studies identified CD203c as a novel therapeutic target on neoplastic MCs, and AGS-16C3F as a promising ADC for the treatment of patients with AdvSM.
Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , Immunoconjugates/therapeutic use , Mastocytosis, Systemic/drug therapy , Animals , Apoptosis/drug effects , Cell Line , Cell Proliferation/drug effects , Humans , Mastocytosis, Systemic/pathology , Mice , Mice, Inbred NOD , Mice, SCIDABSTRACT
INTRODUCTION: The study of mammalian development has offered many insights into the molecular aetiology of cancer. We previously used analysis of mammary morphogenesis to discover a critical role for GATA-3 in mammary developmental and carcinogenesis. In recent years an important role for microRNAs (miRNAs) in a myriad of cellular processes in development and in oncogenesis has emerged. METHODS: microRNA profiling was conducted on stromal and epithelial cellular subsets microdissected from the pubertal mouse mammary gland. miR-184 was reactivated by transient or stable overexpression in breast cancer cell lines and examined using a series of in vitro (proliferation, tumour-sphere and protein synthesis) assays. Orthotopic xenografts of breast cancer cells were used to assess the effect of miR-184 on tumourigenesis as well as distant metastasis. Interactions between miR-184 and its putative targets were assessed by quantitative PCR, microarray, bioinformatics and 3' untranslated region Luciferase reporter assay. The methylation status of primary patient samples was determined by MBD-Cap sequencing. Lastly, the clinical prognostic significance of miR-184 putative targets was assessed using publicly available datasets. RESULTS: A large number of microRNA were restricted in their expression to specific tissue subsets. MicroRNA-184 (miR-184) was exclusively expressed in epithelial cells and markedly upregulated during differentiation of the proliferative, invasive cells of the pubertal terminal end bud (TEB) into ductal epithelial cells in vivo. miR-184 expression was silenced in mouse tumour models compared to non-transformed epithelium and in a majority of breast cancer cell line models. Ectopic reactivation of miR-184 inhibited the proliferation and self-renewal of triple negative breast cancer (TNBC) cell lines in vitro and delayed primary tumour formation and reduced metastatic burden in vivo. Gene expression studies uncovered multi-factorial regulation of genes in the AKT/mTORC1 pathway by miR-184. In clinical breast cancer tissues, expression of miR-184 is lost in primary TNBCs while the miR-184 promoter is methylated in a subset of lymph node metastases from TNBC patients. CONCLUSIONS: These studies elucidate a new layer of regulation in the PI3K/AKT/mTOR pathway with relevance to mammary development and tumour progression and identify miR-184 as a putative breast tumour suppressor.
Subject(s)
Breast Neoplasms/genetics , Gene Expression Profiling , Genes, Tumor Suppressor , Mammary Glands, Animal/metabolism , MicroRNAs/genetics , Sexual Maturation/genetics , Animals , Breast Neoplasms/metabolism , Breast Neoplasms/mortality , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation , Cell Transformation, Neoplastic/genetics , Cluster Analysis , Epigenesis, Genetic , Female , Gene Expression Regulation, Neoplastic , Gene Silencing , Humans , Mice , Neoplasm Metastasis , Prognosis , Proto-Oncogene Proteins c-akt/metabolism , RNA Interference , TOR Serine-Threonine Kinases/metabolismABSTRACT
The Cancer Genome Atlas (TCGA) projects have advanced our understanding of the driver mutations, genetic backgrounds, and key pathways activated across cancer types. Analysis of TCGA datasets have mostly focused on somatic mutations and translocations, with less emphasis placed on gene amplifications. Here we describe a bioinformatics screening strategy to identify putative cancer driver genes amplified across TCGA datasets. We carried out GISTIC2 analysis of TCGA datasets spanning 16 cancer subtypes and identified 486 genes that were amplified in two or more datasets. The list was narrowed to 75 cancer-associated genes with potential "druggable" properties. The majority of the genes were localized to 14 amplicons spread across the genome. To identify potential cancer driver genes, we analyzed gene copy number and mRNA expression data from individual patient samples and identified 42 putative cancer driver genes linked to diverse oncogenic processes. Oncogenic activity was further validated by siRNA/shRNA knockdown and by referencing the Project Achilles datasets. The amplified genes represented a number of gene families, including epigenetic regulators, cell cycle-associated genes, DNA damage response/repair genes, metabolic regulators, and genes linked to the Wnt, Notch, Hedgehog, JAK/STAT, NF-KB and MAPK signaling pathways. Among the 42 putative driver genes were known driver genes, such as EGFR, ERBB2 and PIK3CA. Wild-type KRAS was amplified in several cancer types, and KRAS-amplified cancer cell lines were most sensitive to KRAS shRNA, suggesting that KRAS amplification was an independent oncogenic event. A number of MAP kinase adapters were co-amplified with their receptor tyrosine kinases, such as the FGFR adapter FRS2 and the EGFR family adapters GRB2 and GRB7. The ubiquitin-like ligase DCUN1D1 and the histone methyltransferase NSD3 were also identified as novel putative cancer driver genes. We discuss the patient tailoring implications for existing cancer drug targets and we further discuss potential novel opportunities for drug discovery efforts.
Subject(s)
Computational Biology , Gene Amplification , Genomics , Neoplasms/genetics , Oncogenes/genetics , Cell Transformation, Neoplastic/genetics , Chromosome Mapping , Computational Biology/methods , Databases, Nucleic Acid , Datasets as Topic , Epigenesis, Genetic , Gene Dosage , Gene Expression Regulation, Neoplastic , Genomics/methods , Humans , Intracellular Signaling Peptides and Proteins , MAP Kinase Signaling System , Neoplasms/drug therapy , Proteins , Proto-Oncogene Proteins/geneticsABSTRACT
Transforming growth factor (ß1TGFß1) can promote proliferation in late stage cancers but acts as a tumor suppressor in normal epithelial cells and in early stage cancers. Although, the TGFß pathway has been shown to play a key role in tumorigenesis and metastasis, only a limited number of models have been developed to understand this process. Here, we present a novel model system to discern this paradoxical role of TGFß1 using the MDA-MB-231 (MB-231) cell line. The MB-231 triple-negative breast cancer cell line has been extensively characterized and has been shown to continue to proliferate and undergo epithelial-to-mesenchymal transition (EMT) upon TGFß1 stimulation. We have previously shown by microarray analysis that expression of GATA3 in MB-231 cells results in reprogramming of these cells from a basal to a luminal subtype associated with a reduction of metastasis and tumorigenesis when implanted as xenografts. We now demonstrate that GATA3 overexpression in these cells results in a reduction of TGFß1 response, reversal of EMT, and most importantly, restoration of sensitivity to the inhibitory effects on proliferation of TGFß1. Microarray analysis revealed that TGFß1 treatment resulted in reduction of several cell cycle effectors in 231-GATA3 cells but not in control cells. Furthermore, our microarray analysis revealed a significant increase of BMP5 in 231-GATA3 cells. We demonstrate that combined treatment of MB-231 control cells with TGFß1 and BMP5 results in a significant reduction of cellular proliferation. Thus, this model offers a means to further investigate potentially novel mechanisms involved in the switch in response to TGFß1 from tumor promoter to tumor suppressor through the reprogramming of a triple-negative breast cancer cell line by the GATA3 transcription factor.
Subject(s)
Breast Neoplasms/pathology , GATA3 Transcription Factor/genetics , GATA3 Transcription Factor/metabolism , Gene Expression Regulation, Neoplastic , Transforming Growth Factor beta/pharmacology , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Bone Morphogenetic Protein 5/metabolism , Breast Neoplasms/genetics , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Epithelial-Mesenchymal Transition/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Receptor, ErbB-2/genetics , Receptors, Estrogen/genetics , Receptors, Progesterone/genetics , Signal Transduction/drug effects , Transcription, Genetic/drug effects , Transforming Growth Factor beta/metabolismABSTRACT
Cancer metastases arise in part from disseminated tumor cells originating from the primary tumor and from residual disease persisting after therapy. The identification of biomarkers on micro-metastases, disseminated tumors, and residual disease may yield novel tools for early detection and treatment of these disease states prior to their development into metastases and recurrent tumors. Here we describe the molecular profiling of disseminated tumor cells in lungs, lung metastases, and residual tumor cells in the MMTV-PyMT breast cancer model. MMTV-PyMT mice were bred with actin-GFP mice, and focal hyperplastic lesions from pubertal MMTV-PyMT;actin-GFP mice were orthotopically transplanted into FVB/n mice to track single tumor foci. Tumor-bearing mice were treated with TAC chemotherapy (docetaxel, doxorubicin, cyclophosphamide), and residual and relapsed tumor cells were sorted and profiled by mRNA microarray analysis. Data analysis revealed enrichment of the Jak/Stat pathway, Notch pathway, and epigenetic regulators in residual tumors. Stat1 was significantly up-regulated in a DNA-damage-resistant population of residual tumor cells, and a pre-existing Stat1 sub-population was identified in untreated tumors. Tumor cells from adenomas, carcinomas, lung disseminated tumor cells, and lung metastases were also sorted from MMTV-PyMT transplant mice and profiled by mRNA microarray. Whereas disseminated tumors cells appeared similar to carcinoma cells at the mRNA level, lung metastases were genotypically very different from disseminated cells and primary tumors. Lung metastases were enriched for a number of chromatin-modifying genes and stem cell-associated genes. Histone analysis of H3K4 and H3K9 suggested that lung metastases had been reprogrammed during malignant progression. These data identify novel biomarkers of residual tumor cells and disseminated tumor cells and implicate pathways that may mediate metastasis formation and tumor relapse after therapy.
Subject(s)
Biomarkers, Tumor/biosynthesis , Gene Expression Regulation, Neoplastic , Lung Neoplasms/metabolism , Mammary Neoplasms, Experimental/metabolism , Mammary Tumor Virus, Mouse , Neoplastic Cells, Circulating/metabolism , Animals , Biomarkers, Tumor/genetics , Gene Expression Profiling , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Lung Neoplasms/therapy , Male , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/pathology , Mammary Neoplasms, Experimental/therapy , Mice , Mice, Transgenic , Neoplasm Metastasis , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Neoplasm, Residual , Neoplastic Cells, Circulating/pathology , Oligonucleotide Array Sequence Analysis , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Neoplasm/biosynthesis , RNA, Neoplasm/genetics , STAT Transcription Factors/metabolismABSTRACT
UNLABELLED: The transcription factor ZNF217 is a candidate oncogene in the amplicon on chromosome 20q13 that occurs in 20% to 30% of primary human breast cancers and that correlates with poor prognosis. We show that Znf217 overexpression drives aberrant differentiation and signaling events, promotes increased self-renewal capacity, mesenchymal marker expression, motility, and metastasis, and represses an adult tissue stem cell gene signature downregulated in cancers. By in silico screening, we identified candidate therapeutics that at low concentrations inhibit growth of cancer cells expressing high ZNF217. We show that the nucleoside analogue triciribine inhibits ZNF217-induced tumor growth and chemotherapy resistance and inhibits signaling events [e.g., phospho-AKT, phospho-mitogen-activated protein kinase (MAPK)] in vivo. Our data suggest that ZNF217 is a biomarker of poor prognosis and a therapeutic target in patients with breast cancer and that triciribine may be part of a personalized treatment strategy in patients overexpressing ZNF217. Because ZNF217 is amplified in numerous cancers, these results have implications for other cancers. SIGNIFICANCE: This study finds that ZNF217 is a poor prognostic indicator and therapeutic target in patients with breast cancer and may be a strong biomarker of triciribine treatment efficacy in patients. Because previous clinical trials for triciribine did not include biomarkers of treatment efficacy, this study provides a rationale for revisiting triciribine in the clinical setting as a therapy for patients with breast cancer who overexpress ZNF217.
Subject(s)
Biomarkers, Tumor/genetics , Breast Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Trans-Activators/genetics , Animals , Antibiotics, Antineoplastic/pharmacology , Biomarkers, Tumor/metabolism , Blotting, Western , Breast Neoplasms/pathology , Breast Neoplasms/prevention & control , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Progression , Doxorubicin/pharmacology , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Female , Gene Expression Profiling , Humans , MCF-7 Cells , Mice , NIH 3T3 Cells , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Oligonucleotide Array Sequence Analysis , Prognosis , Reverse Transcriptase Polymerase Chain Reaction , Ribonucleosides/pharmacology , Survival Analysis , Trans-Activators/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Several ongoing clinical studies are designed to test the efficacy of antiangiogenic therapies in the adjuvant setting, where the goal is to increase the cure rate in patients who have just undergone surgical resection of all visible disease. Tumors depend on angiogenesis to support their growth and progression and blockade of this process has proven to be a valid strategy for treating multiple types of advanced metastatic cancer. However, results from the first of these clinical adjuvant studies were disappointing, stimulating extensive debate as to the potential of this approach. It will require additional clinical studies before we realize whether the effects of angiogenic blockade are durable, and if they are able to cure a subset of patients with early stage cancer.
Subject(s)
Angiogenesis Inhibitors/therapeutic use , Antibodies, Monoclonal/therapeutic use , Neoplasms/drug therapy , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal, Humanized , Bevacizumab , Chemotherapy, Adjuvant , Drug Screening Assays, Antitumor , Humans , Neoplasms/blood supply , Vascular Endothelial Growth Factor A/immunologyABSTRACT
The GATA family of transcription factors plays essential roles in the specification and maintenance of differentiated cell types. GATA-3 was identified in a microarray screen of the mouse mammary gland as the most highly expressed transcription factor in the mammary epithelium and is expressed exclusively in the luminal epithelial cell population. Targeted deletion of GATA-3 in mammary glands leads to profound defects in mammary development and inability to specify and maintain the luminal cell fate in the adult mouse. In breast cancer, GATA-3 has emerged as a strong predictor of tumor differentiation, estrogen-receptor status, and clinical outcome. GATA-3 maintains tumor differentiation and suppresses tumor dissemination in a mouse model of breast cancer. This review explores our current understanding of GATA-3 signaling in luminal cell differentiation, both in mammary development and breast cancer.
Subject(s)
Cell Lineage , GATA3 Transcription Factor/metabolism , Mammary Glands, Human/cytology , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , HumansABSTRACT
How breast cancers are able to disseminate and metastasize is poorly understood. Using a hyperplasia transplant system, we show that tumor dissemination and metastasis occur in discrete steps during tumor progression. Bioinformatic analysis revealed that loss of the transcription factor GATA-3 marked progression from adenoma to early carcinoma and onset of tumor dissemination. Restoration of GATA-3 in late carcinomas induced tumor differentiation and suppressed tumor dissemination. Targeted deletion of GATA-3 in early tumors led to apoptosis of differentiated cells, indicating that its loss is not sufficient for malignant conversion. Rather, malignant progression occurred with an expanding GATA-3-negative tumor cell population. These data indicate that GATA-3 regulates tumor differentiation and suppresses tumor dissemination in breast cancer.
Subject(s)
Breast Neoplasms/pathology , Cell Differentiation , GATA3 Transcription Factor/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Animals , Biomarkers, Tumor/metabolism , Breast Neoplasms/genetics , Cell Movement , Cell Proliferation , Disease Models, Animal , Disease Progression , Epithelial Cells/pathology , Female , GATA3 Transcription Factor/deficiency , Gene Deletion , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Hyperplasia , Immunohistochemistry , Mammary Glands, Animal , Mice , Neoplasm Metastasis , Neoplasm Transplantation , Neoplastic Stem Cells/pathologyABSTRACT
E-cadherin (E-cad) is a transmembrane adhesion glycoprotein, the expression of which is often reduced in invasive or metastatic tumors. To assess E-cad's distribution among different types of cancer cells, we used bisulfite-sequencing for detailed, base-by-base measurement of CpG methylation in E-cad's promoter region in the NCI-60 cell lines. The mean methylation levels of the cell lines were distributed bimodally, with values pushed toward either the high or low end of the methylation scale. The 38 epithelial cell lines showed substantially lower (28%) mean methylation levels compared with the nonepithelial cell lines (58%). The CpG site at -143 with respect to the transcriptional start was commonly methylated at intermediate levels, even in cell lines with low overall DNA methylation. We also profiled the NCI-60 cell lines using Affymetrix U133 microarrays and found E-cad expression to be correlated with E-cad methylation at highly statistically significant levels. Above a threshold of approximately 20% to 30% mean methylation, the expression of E-cad was effectively silenced. Overall, this study provides a type of detailed analysis of methylation that can also be applied to other cancer-related genes. As has been shown in recent years, DNA methylation status can serve as a biomarker for use in choosing therapy.
Subject(s)
Cadherins/genetics , DNA Methylation , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic , Base Sequence , Cadherins/metabolism , Cell Line, Tumor , Cluster Analysis , CpG Islands , Gene Expression Regulation, Neoplastic , Gene Silencing , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Analysis, DNA , Sequence Homology, Nucleic AcidABSTRACT
The GATA family of transcription factors plays fundamental roles in cell-fate specification. However, it is unclear if these genes are necessary for the maintenance of cellular differentiation after development. We identified GATA-3 as the most highly enriched transcription factor in the mammary epithelium of pubertal mice. GATA-3 was found in the luminal cells of mammary ducts and the body cells of terminal end buds (TEBs). Upon conditional deletion of GATA-3, mice exhibited severe defects in mammary development due to failure in TEB formation during puberty. After acute GATA-3 loss, adult mice exhibited undifferentiated luminal cell expansion with basement-membrane detachment, which led to caspase-mediated cell death in the long term. Further, FOXA1 was identified as a downstream target of GATA-3 in the mammary gland. This suggests that GATA-3 actively maintains luminal epithelial differentiation in the adult mammary gland, which raises important implications for the pathogenesis of breast cancer.
Subject(s)
Cell Differentiation , Cell Lineage , GATA3 Transcription Factor/metabolism , Mammary Glands, Animal/cytology , Animals , Cell Death , Epithelium/pathology , GATA3 Transcription Factor/deficiency , Hepatocyte Nuclear Factor 3-alpha/genetics , Lactation/physiology , Mammary Glands, Animal/growth & development , Mammary Glands, Animal/pathology , Mice , Mice, Inbred C57BL , Promoter Regions, Genetic/genetics , Time FactorsABSTRACT
The mammary gland develops in a process known as branching morphogenesis, whereby a distal epithelial bud extends and bifurcates to form an extensive ductal network. Compared with other branched organs, such as the lung and kidney, little is known about the molecular basis of branching in the mammary gland. Here we report a microarray profiling strategy to identify novel genes that may regulate mammary branching. We microdissected terminal end bud (TEB) and mature duct microenvironments from beta-actin-green fluorescent protein reporter mice and compared their RNA expression profiles with epithelium-free mammary stroma by means of microarray. We identified 1,074 genes enriched in the TEB microenvironment, 222 genes enriched in the mature duct microenvironment, and 385 genes enriched in both TEB and mature duct microenvironments. The microarray correctly predicted the expression of genes known to be enriched in the epithelium (Ets-5) and stroma (MMP-14) of TEBs and in the mature duct microenvironment (MMP-3). The microarray also correctly predicted the localization of previously uncharacterized genes, such as the TEB-enriched SPRR-1a, the duct-enriched casein-gamma, and the general epithelial marker pleiotrophin. Analysis of genes enriched in TEBs revealed several genes in the Wnt (Wnt-2, Wnt-5a, Wnt-7b, Dsh-3, Frizzled-1, Frizzled-2), hedgehog (Dhh), ephrin (Ephrin-B1, Eph-A2), and transcription factor (Twist-1, Twist-2, Snail) families. In situ hybridization verified that these genes were enriched in the TEB epithelium (Wnt-5a, Wnt-7b, Dhh, Eph-A2) or TEB stroma (Wnt-2, Frizzled-1, Ephrin-B1). We discuss the potential roles of these genes in mammary branching morphogenesis.
Subject(s)
Mammary Glands, Animal/growth & development , Mammary Glands, Animal/metabolism , Animals , Base Sequence , DNA, Complementary/genetics , Ephrins/genetics , Ephrins/metabolism , Female , Gene Expression Profiling , Gene Expression Regulation, Developmental , Genome , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , In Situ Hybridization , Mice , Mice, Transgenic , Morphogenesis/genetics , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic , Wnt Proteins/genetics , Wnt Proteins/metabolismABSTRACT
Unlike other branched organs, the mammary gland undergoes most of its branching during adolescent rather than embryonic development. Its morphogenesis begins in utero, pauses between birth and puberty, and resumes in response to ovarian estrogens to form an open ductal tree that eventually fills the entire mammary fat pad of the young female adult. Importantly, this "open" architecture leaves room during pregnancy for the organ to develop milk-producing alveoli like leaves on otherwise bare branches. Thereafter, the ducts serve to deliver the milk that is produced throughout lactation. The hormonal cues that elicit these various phases of mammary development utilize local signaling cascades and reciprocal stromal-epithelial interactions to orchestrate the tissue reorganization, differentiation and specific activities that define each phase. Fortunately, the mammary gland is rather amenable to experimental inquiry and, as a result, we have a fair, although incomplete, understanding of the mechanisms that control its development. This review discusses our current sense and understanding of those mechanisms as they pertain to mammary branching, with the caveat that many more aspects are still waiting to be solved.
Subject(s)
Breast/growth & development , Morphogenesis , Animals , Breast/embryology , Cell Communication , ErbB Receptors/physiology , Female , Humans , Receptor, ErbB-2/physiology , Signal TransductionABSTRACT
Chromosome rearrangement, a hallmark of cancer, has profound effects on carcinogenesis and tumor phenotype. We used a panel of 60 human cancer cell lines (the NCI-60) as a model system to identify relationships among DNA copy number, mRNA expression level, and drug sensitivity. For each of 64 cancer-relevant genes, we calculated all 4,096 possible Pearson's correlation coefficients relating DNA copy number (assessed by comparative genomic hybridization using bacterial artificial chromosome microarrays) and mRNA expression level (determined using both cDNA and Affymetrix oligonucleotide microarrays). The analysis identified an association of ERBB2 overexpression with 3p copy number, a finding supported by data from human tumors and a mouse model of ERBB2-induced carcinogenesis. When we examined the correlation between DNA copy number for all 353 unique loci on the bacterial artificial chromosome microarray and drug sensitivity for 118 drugs with putatively known mechanisms of action, we found a striking negative correlation (-0.983; 95% bootstrap confidence interval, -0.999 to -0.899) between activity of the enzyme drug L-asparaginase and DNA copy number of genes near asparagine synthetase in the ovarian cancer cells. Previous analysis of drug sensitivity and mRNA expression had suggested an inverse relationship between mRNA levels of asparagine synthetase and L-asparaginase sensitivity in the NCI-60. The concordance of pharmacogenomic findings at the DNA and mRNA levels strongly suggests further study of L-asparaginase for possible treatment of a low-synthetase subset of clinical ovarian cancers. The DNA copy number database presented here will enable other investigators to explore DNA transcript-drug relationships in their own domains of research focus.
Subject(s)
Cell Line, Tumor/drug effects , Cell Line, Tumor/metabolism , DNA, Neoplasm/drug effects , Gene Expression Regulation, Neoplastic , Neoplasm Proteins/genetics , RNA, Messenger/genetics , RNA, Neoplasm/drug effects , Antineoplastic Agents/pharmacology , DNA, Neoplasm/genetics , Humans , Karyotyping , Nucleic Acid Hybridization , Oligonucleotide Array Sequence Analysis , Phenotype , RNA, Messenger/drug effects , RNA, Neoplasm/geneticsABSTRACT
BACKGROUND: Monoclonal antibodies are used extensively throughout the biomedical sciences for detection of antigens, either in vitro or in vivo. We, for example, have used them for quantitation of proteins on "reverse-phase" protein lysate arrays. For those studies, we quality-controlled > 600 available monoclonal antibodies and also needed to develop precise information on the genes that encode their antigens. Translation among the various protein and gene identifier types proved non-trivial because of one-to-many and many-to-one relationships. To organize the antibody, protein, and gene information, we initially developed a relational database in Filemaker for our own use. When it became apparent that the information would be useful to many other researchers faced with the need to choose or characterize antibodies, we developed it further as AbMiner, a fully relational web-based database under MySQL, programmed in Java. DESCRIPTION: AbMiner is a user-friendly, web-based relational database of information on > 600 commercially available antibodies that we validated by Western blot for protein microarray studies. It includes many types of information on the antibody, the immunogen, the vendor, the antigen, and the antigen's gene. Multiple gene and protein identifier types provide links to corresponding entries in a variety of other public databases, including resources for phosphorylation-specific antibodies. AbMiner also includes our quality-control data against a pool of 60 diverse cancer cell types (the NCI-60) and also protein expression levels for the NCI-60 cells measured using our high-density "reverse-phase" protein lysate microarrays for a selection of the listed antibodies. Some other available database resources give information on antibody specificity for one or a couple of cell types. In contrast, the data in AbMiner indicate specificity with respect to the antigens in a pool of 60 diverse cell types from nine different tissues of origin. CONCLUSION: AbMiner is a relational database that provides extensive information from our own laboratory and other sources on more than 600 available antibodies and the genes that encode the antibodies' antigens. The data will be made freely available at http://discover.nci.nih.gov/abminer.
Subject(s)
Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Computational Biology/methods , Databases, Protein , Genomics/methods , Immunologic Techniques , User-Computer Interface , Database Management Systems , Information Storage and Retrieval/methods , Internet , Protein Array Analysis/methods , Proteomics/methods , ResearchABSTRACT
Epithelial-mesenchymal crosstalk is essential for tissue morphogenesis, but incompletely understood. Postnatal mammary gland development requires epidermal growth factor receptor (EGFR) and its ligand amphiregulin (AREG), which generally must be cleaved from its transmembrane form in order to function. As the transmembrane metalloproteinase ADAM17 can process AREG in culture and Adam17(-/-) mice tend to phenocopy Egfr(-/-) mice, we examined the role of each of these molecules in mammary development. Tissue recombination and transplantation studies revealed that EGFR phosphorylation and ductal development occur only when ADAM17 and AREG are expressed on mammary epithelial cells, whereas EGFR is required stromally, and that local AREG administration can rescue Adam17(-/-) transplants. Several EGFR agonists also stimulated Adam17(-/-) mammary organoid growth in culture, but only AREG was expressed abundantly in the developing ductal system in vivo. Thus, ADAM17 plays a crucial role in mammary morphogenesis by releasing AREG from mammary epithelial cells, thereby eliciting paracrine activation of stromal EGFR and reciprocal responses that regulate mammary epithelial development.
Subject(s)
ErbB Receptors/metabolism , Glycoproteins/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Mammary Glands, Animal/embryology , Mammary Glands, Animal/metabolism , Metalloendopeptidases/metabolism , Paracrine Communication , Stromal Cells/metabolism , ADAM Proteins , ADAM17 Protein , Amphiregulin , Animals , Cells, Cultured , EGF Family of Proteins , Epithelial Cells/metabolism , ErbB Receptors/genetics , Female , Gene Expression Profiling , Gene Expression Regulation, Developmental , Glycoproteins/deficiency , Glycoproteins/genetics , Glycoproteins/pharmacology , Intercellular Signaling Peptides and Proteins/deficiency , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/pharmacology , Ligands , Mammary Glands, Animal/growth & development , Metalloendopeptidases/genetics , Mice , Mice, Knockout , Morphogenesis/drug effects , Organoids/metabolism , Phosphorylation , Phosphotyrosine/metabolismABSTRACT
Because most potential molecular markers and targets are proteins, proteomic profiling is expected to yield more direct answers to functional and pharmacological questions than does transcriptional profiling. To aid in such studies, we have developed a protocol for making reverse-phase protein lysate microarrays with larger numbers of spots than previously feasible. Our first application of these arrays was to profiling of the 60 human cancer cell lines (NCI-60) used by the National Cancer Institute to screen compounds for anticancer activity. Each glass slide microarray included 648 lysate spots representing the NCI-60 cell lines plus controls, each at 10 two-fold serial dilutions to provide a wide dynamic range. Mouse monoclonal antibodies and the catalyzed signal amplification system were used for immunoquantitation. The signal levels from the >30,000 data points for our first 52 antibodies were analyzed by using p-scan and a quantitative dose interpolation method. Clustered image maps revealed biologically interpretable patterns of protein expression. Among the principal early findings from these arrays were two promising pathological markers for distinguishing colon from ovarian adenocarcinomas. When we compared the patterns of protein expression with those we had obtained for the same genes at the mRNA level by using both cDNA and oligonucleotide arrays, a striking regularity appeared: cell-structure-related proteins almost invariably showed a high correlation between mRNA and protein levels across the NCI-60 cell lines, whereas non-cell-structure-related proteins showed poor correlation.
Subject(s)
Protein Array Analysis/methods , Proteomics/methods , Blotting, Western , Cell Line, Tumor , Drug Screening Assays, Antitumor/methods , Drug Screening Assays, Antitumor/statistics & numerical data , Female , Gene Expression Profiling , Humans , Male , Neoplasm Proteins/genetics , Protein Array Analysis/statistics & numerical data , Reproducibility of ResultsABSTRACT
To study the molecular mechanisms by which drug resistance develops, we compared DU145 humanprostate cancer cells with a subline selected for resistance to camptothecin. Differences in gene expression level were assessed by hybridizing the two cell types against each other using quadruplicate "Oncochip" cDNA microarrays that included 1648 cancer-related genes. Expression levels differing by a factor of >1.5 were detected for 181 of the genes. These differences were judged statistically reliable on the basis of a stratum-adjusted Kruskal-Wallis test, after taking into account a dye-dependent variable. The 181 expression-altered genes included a larger than expected number of the "apoptosis-related" genes (P = 0.04). To assess whether this observation reflected a generalized resistance of RCO.1 to apoptosis, we exposed the cells to a range of stresses (cisplatin, staurosporine, UV, ionizing radiation, and serum starvation) and found greatly reduced apoptotic responses for RC0.1 (relative to DU145) using flow cytometric Annexin V and terminal deoxynucleotidyl transferase-mediated nick end labeling assays. We next examined the apoptosis-related genes in the context of a molecular interaction map and found expression differences in the direction "expected" on the basis of the apoptosis-resistance of RC0.1 for BAD, caspase-6, and genes that signal via the Akt pathway. Exposure of the cells to wortmannin, an inhibitor of the Akt effector phosphatidylinositol 3-kinase, provided functional support for involvement of the Akt pathway. However, closer examination of the molecular interaction map revealed a paradox: many of the expression differences observed for apoptosis-related genes were in the direction "contrary" to that expected given the resistance of RC0.1. The map indicated that most of these unexpected expression differences were associated with genes involved in the nuclear factor kappa B and transforming growth factor beta pathways. Overall, the patterns that emerged suggested a two-step model for the selection process that led to resistance in RC0.1 cells. The first hypothesized step would involve a decrease in apoptotic susceptibility through changes in the apoptosis-control machinery associated with the Bcl-2 and caspase gene families, and also in antiapoptotic pathways operating through Akt/PKB. The second step would involve changes in multifunctional upstream genes (including some genes in the nuclear factor kappa B and transforming growth factor beta pathways) that can facilitate apoptosis but that would also tend to contribute to cell proliferation in the presence of drug. Thus, we propose that a downstream blockade of apoptosis was "permissive" for the selection of upstream pathway changes that would otherwise have induced apoptosis. This model is analogous to one suggested previously for the relationship between oncogene function and apoptosis in carcinogenesis.