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1.
J Clin Invest ; 133(9)2023 05 01.
Article in English | MEDLINE | ID: mdl-36951942

ABSTRACT

Patients with small cell lung cancer (SCLC) generally have a poor prognosis and a median overall survival of only about 13 months, indicating the urgent need for novel therapies. Delta-like protein 3 (DLL3) has been identified as a tumor-specific cell surface marker on neuroendocrine cancers, including SCLC. In this study, we developed a chimeric antigen receptor (CAR) against DLL3 that displays antitumor efficacy in xenograft and murine SCLC models. CAR T cell expression of the proinflammatory cytokine IL-18 greatly enhanced the potency of DLL3-targeting CAR T cell therapy. In a murine metastatic SCLC model, IL-18 production increased the activation of both CAR T cells and endogenous tumor-infiltrating lymphocytes. We also observed an increased infiltration, repolarization, and activation of antigen-presenting cells. Additionally, human IL-18-secreting anti-DLL3 CAR T cells showed an increased memory phenotype, less exhaustion, and induced durable responses in multiple SCLC models, an effect that could be further enhanced with anti-PD-1 blockade. All together, these results define DLL3-targeting CAR T cells that produce IL-18 as a potentially promising novel strategy against DLL3-expressing solid tumors.


Subject(s)
Carcinoma, Neuroendocrine , Interleukin-18 , Lung Neoplasms , Small Cell Lung Carcinoma , Animals , Humans , Mice , Cell Line, Tumor , Immunotherapy, Adoptive , Interleukin-18/pharmacology , Interleukin-18/therapeutic use , Lung Neoplasms/genetics , Small Cell Lung Carcinoma/genetics , T-Lymphocytes/pathology , Xenograft Model Antitumor Assays
2.
Cancer Discov ; 11(12): 3214-3229, 2021 12 01.
Article in English | MEDLINE | ID: mdl-34344693

ABSTRACT

Small cell lung cancer (SCLC) has limited therapeutic options and an exceptionally poor prognosis. Understanding the oncogenic drivers of SCLC may help define novel therapeutic targets. Recurrent genomic rearrangements have been identified in SCLC, most notably an in-frame gene fusion between RLF and MYCL found in up to 7% of the predominant ASCL1-expressing subtype. To explore the role of this fusion in oncogenesis and tumor progression, we used CRISPR/Cas9 somatic editing to generate a Rlf-Mycl-driven mouse model of SCLC. RLF-MYCL fusion accelerated transformation and proliferation of murine SCLC and increased metastatic dissemination and the diversity of metastatic sites. Tumors from the RLF-MYCL genetically engineered mouse model displayed gene expression similarities with human RLF-MYCL SCLC. Together, our studies support RLF-MYCL as the first demonstrated fusion oncogenic driver in SCLC and provide a new preclinical mouse model for the study of this subtype of SCLC. SIGNIFICANCE: The biological and therapeutic implications of gene fusions in SCLC, an aggressive metastatic lung cancer, are unknown. Our study investigates the functional significance of the in-frame RLF-MYCL gene fusion by developing a Rlf-Mycl-driven genetically engineered mouse model and defining the impact on tumor growth and metastasis. This article is highlighted in the In This Issue feature, p. 2945.


Subject(s)
Lung Neoplasms , Small Cell Lung Carcinoma , Animals , Carcinogenesis/genetics , Cell Line, Tumor , Gene Fusion , Genes, myc , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Mice , Proto-Oncogene Proteins c-myc , Small Cell Lung Carcinoma/genetics , Small Cell Lung Carcinoma/pathology , Telomere-Binding Proteins
3.
Pharmacol Ther ; 178: 83-91, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28342824

ABSTRACT

Chimeric antigen receptor (CAR) T cell therapy has shown promising efficacy against hematologic malignancies. Antitumor activity of CAR T cells, however, needs to be improved to increase therapeutic efficacy in both hematologic and solid cancers. Limitations to overcome are 'on-target, off-tumor' toxicity, antigen escape, short CAR T cell persistence, little expansion, trafficking to the tumor and inhibition of T cell activity by an inhibitory tumor microenvironment. Here we will discuss how optimizing the design of CAR T cells through genetic engineering addresses these limitations and improves the antitumor efficacy of CAR T cell therapy in pre-clinical models.


Subject(s)
Immunotherapy , Neoplasms/therapy , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes/immunology , Animals , Humans , Neoplasms/immunology
4.
Cancer Res ; 76(20): 6084-6094, 2016 10 15.
Article in English | MEDLINE | ID: mdl-27550455

ABSTRACT

The PARP inhibitor AZD2461 was developed as a next-generation agent following olaparib, the first PARP inhibitor approved for cancer therapy. In BRCA1-deficient mouse models, olaparib resistance predominantly involves overexpression of P-glycoprotein, so AZD2461 was developed as a poor substrate for drug transporters. Here we demonstrate the efficacy of this compound against olaparib-resistant tumors that overexpress P-glycoprotein. In addition, AZD2461 was better tolerated in combination with chemotherapy than olaparib in mice, which suggests that AZD2461 could have significant advantages over olaparib in the clinic. However, this superior toxicity profile did not extend to rats. Investigations of this difference revealed a differential PARP3 inhibitory activity for each compound and a higher level of PARP3 expression in bone marrow cells from mice as compared with rats and humans. Our findings have implications for the use of mouse models to assess bone marrow toxicity for DNA-damaging agents and inhibitors of the DNA damage response. Finally, structural modeling of the PARP3-active site with different PARP inhibitors also highlights the potential to develop compounds with different PARP family member specificity profiles for optimal antitumor activity and tolerability. Cancer Res; 76(20); 6084-94. ©2016 AACR.


Subject(s)
Neoplasms, Experimental/drug therapy , Phthalazines/pharmacology , Piperidines/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerases/physiology , ATP Binding Cassette Transporter, Subfamily B, Member 1/physiology , Animals , Bone Marrow/drug effects , Cell Line, Tumor , DNA Damage , DNA Repair , Dacarbazine/administration & dosage , Dacarbazine/analogs & derivatives , Drug Discovery , Genes, BRCA1 , Humans , Mice , Phthalazines/administration & dosage , Phthalazines/toxicity , Piperazines/administration & dosage , Piperidines/toxicity , Poly(ADP-ribose) Polymerases/chemistry , Rats , Temozolomide , Xenograft Model Antitumor Assays
5.
Oncotarget ; 7(39): 63537-63548, 2016 Sep 27.
Article in English | MEDLINE | ID: mdl-27566577

ABSTRACT

Breast cancer arising in female BRCA1 mutation carriers is characterized by an aggressive phenotype and early age of onset. We performed tandem mass spectrometry-based proteomics of secretomes and exosome-like extracellular vesicles from BRCA1-deficient and BRCA1-proficient murine breast tumor models to identify extracellular protein biomarkers, which can be used as an adjunct to current diagnostic modalities in patients with BRCA1-deficient breast cancer. We identified 2,107 proteins, of which 215 were highly enriched in the BRCA1-deficient secretome. We demonstrated that BRCA1-deficient secretome proteins could cluster most human BRCA1- and BRCA2-related breast carcinomas at the transcriptome level. Topoisomerase I (TOP1) and P-cadherin (CDH3) expression was investigated by immunohistochemistry on tissue microarrays of a large panel of 253 human breast carcinomas with and without BRCA1/2 mutations. We showed that expression of TOP1 and CDH3 was significantly increased in human BRCA1-related breast carcinomas relative to sporadic cases (p = 0.002 and p < 0.001, respectively). Multiple logistic regression showed that TOP1 (adjusted odds ratio [OR] 3.75; 95% confidence interval [95% CI], 1.85 - 7.71, p < 0.001) as well as CDH3 positivity (adjusted OR 2.45; 95% CI, 1.08 - 5.49, p = 0.032) were associated with BRCA1/2-related breast carcinomas after adjustment for triple-negative phenotype and age. In conclusion, proteome profiling of secretome using murine breast tumor models is a powerful strategy to identify non-invasive candidate biomarkers of BRCA1-deficient breast cancer. We demonstrate that TOP1 and CDH3 are closely associated to BRCA1-deficient breast cancer. These data merit further investigation for early detection of tumors arising in BRCA1 mutation carriers.


Subject(s)
BRCA1 Protein/deficiency , Biomarkers, Tumor/metabolism , Breast Neoplasms/metabolism , Carcinoma, Ductal, Breast/metabolism , Carcinoma, Lobular/metabolism , Carcinoma, Medullary/metabolism , Proteome/analysis , Animals , BRCA1 Protein/genetics , BRCA2 Protein/deficiency , BRCA2 Protein/genetics , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Carcinoma, Ductal, Breast/genetics , Carcinoma, Ductal, Breast/pathology , Carcinoma, Lobular/genetics , Carcinoma, Lobular/pathology , Carcinoma, Medullary/genetics , Carcinoma, Medullary/pathology , Disease Models, Animal , Female , Humans , Mice , Middle Aged , Mutation , Proteomics/methods , Tumor Cells, Cultured
6.
Proc Natl Acad Sci U S A ; 112(27): 8409-14, 2015 Jul 07.
Article in English | MEDLINE | ID: mdl-26100884

ABSTRACT

Metaplastic breast carcinoma (MBC) is a rare histological breast cancer subtype characterized by mesenchymal elements and poor clinical outcome. A large fraction of MBCs harbor defects in breast cancer 1 (BRCA1). As BRCA1 deficiency sensitizes tumors to DNA cross-linking agents and poly(ADP-ribose) polymerase (PARP) inhibitors, we sought to investigate the response of BRCA1-deficient MBCs to the PARP inhibitor olaparib. To this end, we established a genetically engineered mouse model (GEMM) for BRCA1-deficient MBC by introducing the MET proto-oncogene into a BRCA1-associated breast cancer model, using our novel female GEMM ES cell (ESC) pipeline. In contrast to carcinomas, BRCA1-deficient mouse carcinosarcomas resembling MBC show intrinsic resistance to olaparib caused by increased P-glycoprotein (Pgp) drug efflux transporter expression. Indeed, resistance could be circumvented by using another PARP inhibitor, AZD2461, which is a poor Pgp substrate. These preclinical findings suggest that patients with BRCA1-associated MBC may show poor response to olaparib and illustrate the value of GEMM-ESC models of human cancer for evaluation of novel therapeutics.


Subject(s)
BRCA1 Protein/deficiency , Mammary Neoplasms, Experimental/drug therapy , Phthalazines/pharmacology , Piperazines/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/metabolism , Animals , BRCA1 Protein/genetics , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Carcinosarcoma/drug therapy , Carcinosarcoma/genetics , Carcinosarcoma/metabolism , Disease Models, Animal , Drug Resistance, Neoplasm/genetics , Enzyme Inhibitors/pharmacology , Female , Humans , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/metabolism , Metaplasia , Mice, Inbred C57BL , Mice, Knockout , Poly(ADP-ribose) Polymerases/metabolism , Proto-Oncogene Mas , Survival Analysis
7.
Nature ; 521(7553): 541-544, 2015 May 28.
Article in English | MEDLINE | ID: mdl-25799992

ABSTRACT

Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway. In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases. In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.


Subject(s)
DNA Breaks, Double-Stranded , Mad2 Proteins/metabolism , Poly(ADP-ribose) Polymerase Inhibitors , Recombinational DNA Repair , Adaptor Proteins, Signal Transducing , Animals , Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors , Ataxia Telangiectasia Mutated Proteins/metabolism , BRCA1 Protein/deficiency , BRCA1 Protein/genetics , BRCA1 Protein/metabolism , Cell Cycle Proteins , Cell Line , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins/metabolism , Drug Resistance, Neoplasm/genetics , Histones/metabolism , Humans , Immunoglobulin Class Switching/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mad2 Proteins/deficiency , Mad2 Proteins/genetics , Mice , Nuclear Proteins/metabolism , Trans-Activators/metabolism , Tumor Suppressor p53-Binding Protein 1 , Ubiquitin-Protein Ligases/metabolism
8.
Cancer Res ; 75(4): 732-41, 2015 Feb 15.
Article in English | MEDLINE | ID: mdl-25511378

ABSTRACT

Pan- or multidrug resistance is a central problem in clinical oncology. Here, we use a genetically engineered mouse model of BRCA2-associated hereditary breast cancer to study drug resistance to several types of chemotherapy and PARP inhibition. We found that multidrug resistance was strongly associated with an EMT-like sarcomatoid phenotype and high expression of the Abcb1b gene, which encodes the drug efflux transporter P-glycoprotein. Inhibition of P-glycoprotein could partly resensitize sarcomatoid tumors to the PARP inhibitor olaparib, docetaxel, and doxorubicin. We propose that multidrug resistance is a multifactorial process and that mouse models are useful to unravel this.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B/genetics , BRCA2 Protein/genetics , Drug Resistance, Multiple/genetics , Mammary Neoplasms, Animal/genetics , ATP Binding Cassette Transporter, Subfamily B/biosynthesis , Animals , Antineoplastic Agents/administration & dosage , BRCA2 Protein/deficiency , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Doxorubicin/administration & dosage , Drug Resistance, Neoplasm/genetics , Enzyme Inhibitors/administration & dosage , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mammary Neoplasms, Animal/drug therapy , Mammary Neoplasms, Animal/pathology , Mice , Poly(ADP-ribose) Polymerase Inhibitors
9.
Transl Oncol ; 7(2): 230-9, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24726234

ABSTRACT

INTRODUCTION: Anatomic imaging alone is often inadequate for tuning systemic treatment for individual tumor response. Optically based techniques could potentially contribute to fast and objective response monitoring in personalized cancer therapy. In the present study, we evaluated the feasibility of dual-modality diffuse reflectance spectroscopy-autofluorescence spectroscopy (DRS-AFS) to monitor the effects of systemic treatment in a mouse model for hereditary breast cancer. METHODS: Brca1(-/-); p53(-/-) mammary tumors were grown in 36 mice, half of which were treated with a single dose of cisplatin. Changes in the tumor physiology and morphology were measured for a period of 1 week using dual-modality DRS-AFS. Liver and muscle tissues were also measured to distinguish tumor-specific alterations from systemic changes. Model-based analyses were used to derive different optical parameters like the scattering and absorption coefficients, as well as sources of intrinsic fluorescence. Histopathologic analysis was performed for cross-validation with trends in optically based parameters. RESULTS: Treated tumors showed a significant decrease in Mie-scattering slope and Mie-to-total scattering fraction and an increase in both fat volume fraction and tissue oxygenation after 2 days of follow-up. Additionally, significant tumor-specific changes in the fluorescence spectra were seen. These longitudinal trends were consistent with changes observed in the histopathologic analysis, such as vital tumor content and formation of fibrosis. CONCLUSIONS: This study demonstrates that dual-modality DRS-AFS provides quantitative functional information that corresponds well with the degree of pathologic response. DRS-AFS, in conjunction with other imaging modalities, could be used to optimize systemic cancer treatment on the basis of early individual tumor response.

10.
PLoS One ; 8(3): e60357, 2013.
Article in English | MEDLINE | ID: mdl-23555959

ABSTRACT

BACKGROUND: Circulating Endothelial Progenitor Cell (EPC) levels are reduced in diabetes mellitus. This may be a consequence of impaired mobilization of EPC from the bone marrow. We hypothesized that under diabetic conditions, mobilization of EPC from the bone marrow to the circulation is impaired -at least partly- due to dysfunction of the bone marrow stromal compartment. METHODS: Diabetes was induced in mice by streptozotocin injection. Circulating Sca-1(+)Flk-1(+) EPC were characterized and quantified by flow cytometry at baseline and after mobilization with G-CSF/SCF injections. In vivo hemangiogenic recovery was tested by 5-FU challenge. Interaction within the bone marrow environment between CD34(+) hematopoietic progenitor cells (HPC) and supporting stroma was assessed by co-cultures. To study progenitor cell-endothelial cell interaction under normoglycemic and hyperglycemic conditions, a co-culture model using E4Orf1-transfected human endothelial cells was employed. RESULTS: In diabetic mice, bone marrow EPC levels were unaffected. However, circulating EPC levels in blood were lower at baseline and mobilization was attenuated. Diabetic mice failed to recover and repopulate from 5-FU injection. In vitro, primary cultured bone marrow stroma from diabetic mice was impaired in its capacity to support human CFU-forming HPC. Finally, hyperglycemia hampered the HPC supportive function of endothelial cells in vitro. CONCLUSION: EPC mobilization is impaired under experimental diabetic conditions and our data suggest that diabetes induces alterations in the progenitor cell supportive capacity of the bone marrow stroma, which could be partially responsible for the attenuated EPC mobilization and reduced EPC levels observed in diabetic patients.


Subject(s)
Bone Marrow/physiopathology , Diabetes Mellitus, Experimental/physiopathology , Endothelial Cells/pathology , Hematopoietic Stem Cells/pathology , Stem Cells/pathology , Animals , Antigens, CD34/analysis , Cell Count , Cell Movement , Cell Survival , Cells, Cultured , Coculture Techniques , Fluorouracil/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Hyperglycemia/physiopathology , Mice , Mice, Inbred C57BL
11.
Mol Cell Proteomics ; 12(5): 1319-34, 2013 May.
Article in English | MEDLINE | ID: mdl-23397111

ABSTRACT

In contrast to various signatures that predict the prognosis of breast cancer patients, markers that predict chemotherapy response are still elusive. To detect such predictive biomarkers, we investigated early changes in protein expression using two mouse models for distinct breast cancer subtypes who have a differential knock-out status for the breast cancer 1, early onset (Brca1) gene. The proteome of cisplatin-sensitive BRCA1-deficient mammary tumors was compared with that of cisplatin-resistant mammary tumors resembling pleomorphic invasive lobular carcinoma. The analyses were performed 24 h after administration of the maximum tolerable dose of cisplatin. At this time point, drug-sensitive BRCA1-deficient tumors showed DNA damage, but cells were largely viable. By applying paired statistics and quantitative filtering, we identified highly discriminatory markers for the sensitive and resistant model. Proteins up-regulated in the sensitive model are involved in centrosome organization, chromosome condensation, homology-directed DNA repair, and nucleotide metabolism. Major discriminatory markers that were up-regulated in the resistant model were predominantly involved in fatty acid metabolism, such as fatty-acid synthase. Specific inhibition of fatty-acid synthase sensitized resistant cells to cisplatin. Our data suggest that exploring the functional link between the DNA damage response and cancer metabolism shortly after the initial treatment may be a useful strategy to predict the efficacy of cisplatin.


Subject(s)
Antineoplastic Agents/pharmacology , Biomarkers, Tumor/metabolism , Cisplatin/pharmacology , Drug Resistance, Neoplasm , Mammary Neoplasms, Experimental/metabolism , Animals , Biosynthetic Pathways , Cdh1 Proteins/genetics , Fatty Acid Synthase, Type I/genetics , Fatty Acid Synthase, Type I/metabolism , Fatty Acids/biosynthesis , Female , Gene Knockdown Techniques , Genes, BRCA1 , Genes, p53 , Mammary Neoplasms, Experimental/drug therapy , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Knockout , Protein Interaction Maps , Proteome/metabolism , Proteomics , Signal Transduction , Tumor Cells, Cultured
12.
Cancer Discov ; 3(1): 68-81, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23103855

ABSTRACT

UNLABELLED: Inhibition of PARP is a promising therapeutic strategy for homologous recombination-deficient tumors, such as BRCA1-associated cancers. We previously reported that BRCA1-deficient mouse mammary tumors may acquire resistance to the clinical PARP inhibitor (PARPi) olaparib through activation of the P-glycoprotein drug efflux transporter. Here, we show that tumor-specific genetic inactivation of P-glycoprotein increases the long-term response of BRCA1-deficient mouse mammary tumors to olaparib, but these tumors eventually developed PARPi resistance. In a fraction of cases, this resistance is caused by partial restoration of homologous recombination due to somatic loss of 53BP1. Importantly, PARPi resistance was minimized by long-term treatment with the novel PARP inhibitor AZD2461, which is a poor P-glycoprotein substrate. Together, our data suggest that restoration of homologous recombination is an important mechanism for PARPi resistance in BRCA1-deficient mammary tumors and that the risk of relapse of BRCA1-deficient tumors can be effectively minimized by using optimized PARP inhibitors. SIGNIFICANCE: In this study, we show that loss of 53BP1 causes resistance to PARP inhibition in mouse mammary tumors that are deficient in BRCA1. We hypothesize that low expression or absence of 53BP1 also reduces the response of patients with BRCA1-deficient tumors to PARP inhibitors.


Subject(s)
Antineoplastic Agents/therapeutic use , Chromosomal Proteins, Non-Histone/genetics , DNA-Binding Proteins/genetics , Drug Resistance, Neoplasm , Enzyme Inhibitors/therapeutic use , Phthalazines/therapeutic use , Piperazines/therapeutic use , Piperidines/therapeutic use , Poly(ADP-ribose) Polymerase Inhibitors , ATP Binding Cassette Transporter, Subfamily B, Member 1/deficiency , Animals , BRCA1 Protein/genetics , Cell Line, Tumor , DNA Damage , Female , Mammary Neoplasms, Animal/drug therapy , Mice , Mutation , Tumor Suppressor p53-Binding Protein 1
13.
Cancer Res ; 72(9): 2350-61, 2012 May 01.
Article in English | MEDLINE | ID: mdl-22396490

ABSTRACT

The lack of markers to predict chemotherapy responses in patients poses a major handicap in cancer treatment. We searched for gene expression patterns that correlate with docetaxel or cisplatin response in a mouse model for breast cancer associated with BRCA1 deficiency. Array-based expression profiling did not identify a single marker gene predicting docetaxel response, despite an increase in Abcb1 (P-glycoprotein) expression that was sufficient to explain resistance in several poor responders. Intertumoral heterogeneity explained the inability to identify a predictive gene expression signature for docetaxel. To address this problem, we used a novel algorithm designed to detect differential gene expression in a subgroup of the poor responders that could identify tumors with increased Abcb1 transcript levels. In contrast, standard analytical tools, such as significance analysis of microarrays, detected a marker only if it correlated with response in a substantial fraction of tumors. For example, low expression of the Xist gene correlated with cisplatin hypersensitivity in most tumors, and it also predicted long recurrence-free survival of HER2-negative, stage III breast cancer patients treated with intensive platinum-based chemotherapy. Our findings may prove useful for selecting patients with high-risk breast cancer who could benefit from platinum-based therapy.


Subject(s)
Antineoplastic Agents/pharmacology , BRCA1 Protein/deficiency , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Mammary Neoplasms, Experimental/drug therapy , Mammary Neoplasms, Experimental/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , ATP Binding Cassette Transporter, Subfamily B, Member 1/deficiency , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , BRCA1 Protein/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cisplatin/pharmacology , Docetaxel , Female , Gene Expression Profiling , Mammary Neoplasms, Experimental/metabolism , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Transgenic , Randomized Controlled Trials as Topic , Taxoids/pharmacology
14.
Mol Cell Proteomics ; 11(7): M111.013334, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22366898

ABSTRACT

Breast cancer 1, early onset (BRCA1) hereditary breast cancer, a type of cancer with defects in the homology-directed DNA repair pathway, would benefit from the identification of proteins for diagnosis, which might also be of potential use as screening, prognostic, or predictive markers. Sporadic breast cancers with defects in the BRCA1 pathway might also be diagnosed. We employed proteomics based on one-dimensional gel electrophoresis in combination with nano-LC-MS/MS and spectral counting to compare the protein profiles of mammary tumor tissues of genetic mouse models either deficient or proficient in BRCA1. We identified a total of 3,545 proteins, of which 801 were significantly differentially regulated between the BRCA1-deficient and -proficient breast tumors. Pathway and protein complex analysis identified DNA repair and related functions as the major processes associated with the up-regulated proteins in the BRCA1-deficient tumors. In addition, by selecting highly connected nodes, we identified a BRCA1 deficiency signature of 45 proteins that enriches for homology-directed DNA repair deficiency in human gene expression breast cancer data sets. This signature also exhibits prognostic power across multiple data sets, with optimal performance in a data set enriched in tumors deficient in homology-directed DNA repair. In conclusion, by comparing mouse proteomes from BRCA1-proficient and -deficient mammary tumors, we were able to identify several markers associated with BRCA1 deficiency and a prognostic signature for human breast cancer deficient in homology-directed DNA repair.


Subject(s)
BRCA1 Protein/genetics , Biomarkers, Tumor/genetics , Breast Neoplasms/genetics , DNA Repair , Mammary Neoplasms, Animal/genetics , Neoplasm Proteins/genetics , Animals , BRCA1 Protein/deficiency , Breast Neoplasms/diagnosis , Breast Neoplasms/mortality , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Mammary Neoplasms, Animal/diagnosis , Mice , Microarray Analysis , Multigene Family , Mutation , Protein Interaction Mapping , Proteome , Proteomics , Sequence Homology, Amino Acid , Survival Analysis , Tandem Mass Spectrometry
15.
Cancer Res ; 70(15): 6268-76, 2010 Aug 01.
Article in English | MEDLINE | ID: mdl-20631063

ABSTRACT

Familial breast and ovarian cancers are often defective in homologous recombination (HR) due to mutations in the BRCA1 or BRCA2 genes. Cisplatin chemotherapy or poly(ADP-ribose) polymerase (PARP) inhibitors were tested for these tumors in clinical trials. In a screen for novel drugs that selectively kill BRCA2-defective cells, we identified 6-thioguanine (6TG), which induces DNA double-strand breaks (DSB) that are repaired by HR. Furthermore, we show that 6TG is as efficient as a PARP inhibitor in selectively killing BRCA2-defective tumors in a xenograft model. Spontaneous BRCA1-defective mammary tumors gain resistance to PARP inhibitors through increased P-glycoprotein expression. Here, we show that 6TG efficiently kills such BRCA1-defective PARP inhibitor-resistant tumors. We also show that 6TG could kill cells and tumors that have gained resistance to PARP inhibitors or cisplatin through genetic reversion of the BRCA2 gene. Although HR is reactivated in PARP inhibitor-resistant BRCA2-defective cells, it is not fully restored for the repair of 6TG-induced lesions. This is likely to be due to several recombinogenic lesions being formed after 6TG. We show that BRCA2 is also required for survival from mismatch repair-independent lesions formed by 6TG, which do not include DSBs. This suggests that HR is involved in the repair of 6TG-induced DSBs as well as mismatch repair-independent 6TG-induced DNA lesion. Altogether, our data show that 6TG efficiently kills BRCA2-defective tumors and suggest that 6TG may be effective in the treatment of advanced tumors that have developed resistance to PARP inhibitors or platinum-based chemotherapy.


Subject(s)
BRCA2 Protein/deficiency , Colonic Neoplasms/drug therapy , Enzyme Inhibitors/pharmacology , Mammary Neoplasms, Experimental/drug therapy , Poly(ADP-ribose) Polymerase Inhibitors , Thioguanine/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , Animals , Antimetabolites, Antineoplastic/pharmacology , Apoptosis Regulatory Proteins , BRCA2 Protein/genetics , BRCA2 Protein/metabolism , Base Pair Mismatch , Colonic Neoplasms/enzymology , Colonic Neoplasms/genetics , DNA Repair , Drug Synergism , Genes, BRCA2 , HCT116 Cells , Humans , Mammary Neoplasms, Experimental/enzymology , Mammary Neoplasms, Experimental/genetics , Mice
16.
Mol Cancer Ther ; 9(6): 1525-35, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20501804

ABSTRACT

Tyrosine kinase inhibitors (TKI) targeting angiogenesis via inhibition of the vascular endothelial growth factor pathway have changed the medical management of metastatic renal cell carcinoma. Although treatment with TKIs has shown clinical benefit, these drugs will eventually fail patients. The potential mechanisms of resistance to TKIs are poorly understood. To address this question, we obtained an excisional biopsy of a skin metastasis from a patient with clear cell renal carcinoma who initially had a response to sunitinib and eventually progressed with therapy. Tumor pieces were grafted s.c. in athymic nude mice. Established xenografts were treated with sunitinib. Tumor size, microvascular density, and pericyte coverage were determined. Plasma as well as tissue levels for sunitinib were assessed. A tumor-derived cell line was established and assessed in vitro for potential direct antitumor effects of sunitinib. To our surprise, xenografts from the patient who progressed on sunitinib regained sensitivity to the drug. At a dose of 40 mg/kg, sunitinib caused regression of the subcutaneous tumors. Histology showed a marked reduction in microvascular density and pericyte dysfunction. More interestingly, histologic examination of the original skin metastasis revealed evidence of epithelial to mesenchymal transition, whereas the xenografts showed reversion to the clear cell phenotype. In vitro studies showed no inhibitory effect on tumor cell growth at pharmacologically relevant concentrations. In conclusion, the histologic examination in this xenograft study suggests that reversible epithelial to mesenchymal transition may be associated with acquired tumor resistance to TKIs in patients with clear cell renal carcinoma.


Subject(s)
Carcinoma, Renal Cell/drug therapy , Drug Resistance, Neoplasm , Epithelium/pathology , Indoles/therapeutic use , Kidney Neoplasms/drug therapy , Mesoderm/pathology , Pyrroles/therapeutic use , Xenograft Model Antitumor Assays , Angiogenesis Inhibitors/blood , Angiogenesis Inhibitors/pharmacology , Angiogenesis Inhibitors/therapeutic use , Animals , Carcinoma, Renal Cell/blood supply , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/pathology , Cell Line, Tumor , Disease Progression , Drug Resistance, Neoplasm/drug effects , Epithelium/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Indoles/blood , Indoles/pharmacology , Kidney Neoplasms/blood supply , Kidney Neoplasms/genetics , Kidney Neoplasms/pathology , Mesoderm/drug effects , Mice , Mice, Nude , Microvessels/drug effects , Pericytes/drug effects , Pericytes/pathology , Phenotype , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Pyrroles/blood , Pyrroles/pharmacology , Skin Neoplasms/secondary , Sunitinib , Von Hippel-Lindau Tumor Suppressor Protein/genetics
17.
Cancer Res ; 70(4): 1700-10, 2010 Feb 15.
Article in English | MEDLINE | ID: mdl-20145144

ABSTRACT

There is no tailored therapy yet for human basal-like mammary carcinomas. However, BRCA1 dysfunction is frequently present in these malignancies, compromising homology-directed DNA repair. This defect may serve as the tumor's Achilles heel and make the tumor hypersensitive to DNA breaks. We have evaluated this putative synthetic lethality in a genetically engineered mouse model for BRCA1-associated breast cancer, using the topoisomerase I (Top1) poison topotecan as monotherapy and in combination with poly(ADP-ribose) polymerase inhibition by olaparib. All 20 tumors tested were topotecan sensitive, but response heterogeneity was substantial. Although topotecan increased mouse survival, all tumors eventually acquired resistance. As mechanisms of in vivo resistance, we identified overexpression of Abcg2/Bcrp and markedly reduced protein levels of the drug target Top1 (without altered mRNA levels). Tumor-specific genetic ablation of Abcg2 significantly increased overall survival of topotecan-treated animals (P < 0.001), confirming the in vivo relevance of ABCG2 for topotecan resistance in a novel approach. Despite the lack of ABCG2, a putative tumor-initiating cell marker, none of the 11 Abcg2(-/-);Brca1(-/-);p53(-/-) tumors were eradicated, not even by the combination topotecan-olaparib. We find that olaparib substantially increases topotecan toxicity in this model, and we suggest that this might also happen in humans.


Subject(s)
Carcinoma/drug therapy , Drug Resistance, Neoplasm/genetics , Genes, BRCA1/physiology , Genes, p53/physiology , Mammary Neoplasms, Animal/drug therapy , Topotecan/therapeutic use , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily B, Member 1 , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/genetics , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/therapeutic use , Carcinoma/genetics , Carcinoma/pathology , Doxorubicin/therapeutic use , Drug Evaluation, Preclinical , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/therapeutic use , Female , Gene Expression Regulation, Neoplastic/drug effects , Mammary Neoplasms, Animal/genetics , Mammary Neoplasms, Animal/pathology , Maximum Tolerated Dose , Mice , Mice, Knockout , Phthalazines/pharmacology , Phthalazines/therapeutic use , Piperazines/pharmacology , Piperazines/therapeutic use , Topoisomerase I Inhibitors , Topotecan/administration & dosage
18.
Biochim Biophys Acta ; 1796(2): 266-80, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19616605

ABSTRACT

Breast cancer is the most common malignancy among women in developed countries, affecting more than a million women per year worldwide. Over the last decades, our increasing understanding of breast cancer biology has led to the development of endocrine agents against hormone receptor-positive tumors and targeted therapeutics against HER2-expressing tumors. However, no targeted therapy is available for patients with triple-negative breast cancer, lacking expression of hormone receptors and HER2. Overlap between BRCA1-mutated breast cancers and triple-negative tumors suggests that an important part of the triple-negative tumors may respond to therapeutics targeting BRCA1-deficient cells. Here, we review the features shared between triple-negative, basal-like and BRCA1-related breast cancers. We also discuss the development of novel therapeutic strategies to target BRCA1-mutated tumors and triple-negative tumors with BRCA1-like features. Finally, we highlight the utility of mouse models for BRCA1-mutated breast cancer to optimize (combination) therapy and to understand drug resistance.


Subject(s)
Breast Neoplasms/chemistry , Breast Neoplasms/drug therapy , Receptor, ErbB-2/analysis , Receptors, Estrogen/analysis , Receptors, Progesterone/analysis , Recombination, Genetic , Animals , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Cycle , DNA Repair , Disease Models, Animal , Drug Resistance, Neoplasm , Female , Genes, BRCA1 , Humans , Mice , Mutation , Poly(ADP-ribose) Polymerase Inhibitors , Protein Kinase Inhibitors/therapeutic use
19.
Proc Natl Acad Sci U S A ; 105(44): 17079-84, 2008 Nov 04.
Article in English | MEDLINE | ID: mdl-18971340

ABSTRACT

Whereas target-specific drugs are available for treating ERBB2-overexpressing and hormone receptor-positive breast cancers, no tailored therapy exists for hormone receptor- and ERBB2-negative ("triple-negative") mammary carcinomas. Triple-negative tumors account for 15% of all breast cancers and frequently harbor defects in DNA double-strand break repair through homologous recombination (HR), such as BRCA1 dysfunction. The DNA-repair defects characteristic of BRCA1-deficient cells confer sensitivity to poly(ADP-ribose) polymerase 1 (PARP1) inhibition, which could be relevant to treatment of triple-negative tumors. To evaluate PARP1 inhibition in a realistic in vivo setting, we tested the PARP inhibitor AZD2281 in a genetically engineered mouse model (GEMM) for BRCA1-associated breast cancer. Treatment of tumor-bearing mice with AZD2281 inhibited tumor growth without signs of toxicity, resulting in strongly increased survival. Long-term treatment with AZD2281 in this model did result in the development of drug resistance, caused by up-regulation of Abcb1a/b genes encoding P-glycoprotein efflux pumps. This resistance to AZD2281 could be reversed by coadministration of the P-glycoprotein inhibitor tariquidar. Combination of AZD2281 with cisplatin or carboplatin increased the recurrence-free and overall survival, suggesting that AZD2281 potentiates the effect of these DNA-damaging agents. Our results demonstrate in vivo efficacy of AZD2281 against BRCA1-deficient breast cancer and illustrate how GEMMs of cancer can be used for preclinical evaluation of novel therapeutics and for testing ways to overcome or circumvent therapy resistance.


Subject(s)
Antineoplastic Agents/therapeutic use , BRCA1 Protein/genetics , Mammary Neoplasms, Animal/drug therapy , Phthalazines/therapeutic use , Piperazines/therapeutic use , Platinum Compounds/therapeutic use , Poly(ADP-ribose) Polymerase Inhibitors , Animals , Aromatase Inhibitors/therapeutic use , Cisplatin/therapeutic use , DNA Damage , DNA Repair/drug effects , Drug Resistance, Neoplasm/genetics , Female , Mammary Neoplasms, Animal/genetics , Mammary Neoplasms, Animal/pathology , Mice , Mice, Transgenic , Neoplasm Transplantation , Poly(ADP-ribose) Polymerases/genetics
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