Perturbation of Rb, p53, and Brca1 or Brca2 Cooperate in Inducing Metastatic Serous Epithelial Ovarian Cancer

The majority of human high grade serous epithelial ovarian cancer (SEOC) is characterized by frequent mutations in p53 and alterations in the RB and FOXM1 pathways. A subset of human SEOC harbors a combination of germline and somatic mutations as well as epigenetic dysfunction for BRCA1/2. Using Cre-conditional alleles and intrabursal induction by Cre-expressing adenovirus in genetically engineered mice, we analyzed the roles of pathway perturbations in epithelial ovarian cancer initiation and progression. Inactivation of RB-mediated tumor suppression induced surface epithelial proliferation with progression to stage I carcinoma. Additional biallelic inactivation and/or missense p53 mutation in the presence or absence of Brca1/2 caused progression to stage IV disease. As in human SEOC, mice developed peritoneal carcinomatosis, ascites, and distant metastases. Unbiased gene expression and metabolomic profiling confirmed that Rb, p53, and Brca1/2-triple mutant tumors aligned with human SEOC, and not with other intraperitoneal cancers. Together, our findings provide a novel resource for evaluating disease etiology and biomarkers, therapeutic evaluation, and improved imaging strategies in epithelial ovarian cancer

Phenotype-driven chemical screening in zebrafish for compounds that inhibit collective cell migration identifies multiple pathways potentially involved in metastatic invasion

In the last decade, high-throughput chemical screening has become the dominant approach for discovering novel compounds with therapeutic properties. Automated screening using in vitro or cultured cell assays have yielded thousands of candidate drugs for a variety of biological targets, but these approaches have not resulted in an increase in drug discovery despite major increases in expenditures. In contrast, phenotype-driven screens have shown a much stronger success rate, which is why we developed an in vivo assay using transgenic zebrafish with a GFP-marked migrating posterior lateral line primordium (PLLp) to identify compounds that influence collective cell migration. We then conducted a high-throughput screen using a compound library of 2160 annotated bioactive synthetic compounds and 800 natural products to identify molecules that block normal PLLp migration. We identified 165 compounds that interfere with primordium migration without overt toxicity in vivo. Selected compounds were confirmed in their migration-blocking activity by using additional assays for cell migration. We then proved the screen to be successful in identifying anti-metastatic compounds active in vivo by performing orthotopic tumor implantation assays in mice. We demonstrated that the Src inhibitor SU6656, identified in our screen, can be used to suppress the metastatic capacity of a highly aggressive mammary tumor cell line. Finally, we used CRISPR/Cas9-targeted mutagenesis in zebrafish to genetically validate predicted targets of compounds. This approach demonstrates that the migrating PLLp in zebrafish can be used for large-scale, high-throughput screening for compounds that inhibit collective cell migration and, potentially, anti-metastatic compounds

Perturbation of Rb, p53, and Brca1 or Brca2 Cooperate in Inducing Metastatic Serous Epithelial Ovarian Cancer

The majority of human high grade serous epithelial ovarian cancer (SEOC) is characterized by frequent mutations in p53 and alterations in the RB and FOXM1 pathways. A subset of human SEOC harbors a combination of germline and somatic mutations as well as epigenetic dysfunction for BRCA1/2. Using Cre-conditional alleles and intrabursal induction by Cre-expressing adenovirus in genetically engineered mice, we analyzed the roles of pathway perturbations in epithelial ovarian cancer initiation and progression. Inactivation of RB-mediated tumor suppression induced surface epithelial proliferation with progression to stage I carcinoma. Additional biallelic inactivation and/or missense p53 mutation in the presence or absence of Brca1/2 caused progression to stage IV disease. As in human SEOC, mice developed peritoneal carcinomatosis, ascites, and distant metastases. Unbiased gene expression and metabolomic profiling confirmed that Rb, p53, and Brca1/2-triple mutant tumors aligned with human SEOC, and not with other intraperitoneal cancers. Together, our findings provide a novel resource for evaluating disease etiology and biomarkers, therapeutic evaluation, and improved imaging strategies in epithelial ovarian cancer

Aspirin insensitive thrombophilia: Transcript profiling of blood identifies platelet abnormalities and HLA restriction

Aspirin is the most widely used antiplatelet agent because it is safe, efficient, and inexpensive. However, a significant subset of patients does not exhibit a full inhibition of platelet aggregation, termed ‘aspirin resistance’ (AR). Several major studies have observed that AR patients have a 4-fold increased risk of myocardial infarction (MI), stroke, and other thrombotic events. Arachidonic acid-stimulated whole blood aggregation was tested in 132 adults at risk for ischemic events, and identified an inadequate response to aspirin therapy in 9 patients (6.8%). Expression profiling of blood RNA by microarray was used to generate new hypotheses about the etiology of AR. Among the differentially expressed genes, there were decreases in several known platelet transcripts, including clusterin (CLU), glycoproteins IIb/IIIa (ITGA2B/3), lipocalin (LCN2), lactoferrin (LTF), and the thrombopoetin receptor (MPL), but with increased mRNA for the T-cell Th1 chemokine CXCL10. There was a strong association of AR with expression of HLA-DRB4 and HLA-DQA1. Similar HLA changes have been linked to autoimmune disorders, particularly antiphospholipid syndrome (APS), in which autoantibodies to phospholipid/protein complexes can trigger platelet activation. Consistent with APS, AR patients exhibited a 30% reduction in platelet counts. Follow-up testing for autoimmune antibodies observed only borderline titers in AR patients. Overall, these results suggest that AR may be related to changes in platelet gene expression creating a hyperreactive platelet, despite antiplatelet therapy. Future studies will focus on determining the protein levels of these differential transcripts in platelets, and the possible involvement of HLA restriction as a contributing factor

Pathway-Specific Engineered Mouse Allograft Models Functionally Recapitulate Human Serous Epithelial Ovarian Cancer

<div><p>The high mortality rate from ovarian cancers can be attributed to late-stage diagnosis and lack of effective treatment. Despite enormous effort to develop better targeted therapies, platinum-based chemotherapy still remains the standard of care for ovarian cancer patients, and resistance occurs at a high rate. One of the rate limiting factors for translation of new drug discoveries into clinical treatments has been the lack of suitable preclinical cancer models with high predictive value. We previously generated genetically engineered mouse (GEM) models based on perturbation of <i>Tp53</i> and <i>Rb</i> with or without <i>Brca1</i> or <i>Brca2</i> that develop serous epithelial ovarian cancer (SEOC) closely resembling the human disease on histologic and molecular levels. Here, we describe an adaptation of these GEM models to orthotopic allografts that uniformly develop tumors with short latency and are ideally suited for routine preclinical studies. Ovarian tumors deficient in <i>Brca1</i> respond to treatment with cisplatin and olaparib, a PARP inhibitor, whereas <i>Brca1</i>-wild type tumors are non-responsive to treatment, recapitulating the relative sensitivities observed in patients. These mouse models provide the opportunity for evaluation of effective therapeutics, including prediction of differential responses in <i>Brca1</i>-wild type and <i>Brca1</i>–deficient tumors and development of relevant biomarkers.</p></div

Summary of orthotopic syngeneic tumor transplants.

<p>*latency of tumor development from induction or transplantation till end point.</p><p><i>TgK18G_T121^tg/+  = </i> transgenic for bacterial artificial chromosome containing the mouse cytokeratin 18 gene, into which a Cre-conditional loxP-GFP-stop-loxP T₁₂₁ cassette was inserted.</p><p><i>Brca1^Δ/Δ  = </i> deletion mutant for <i>Brca1</i> gene.</p><p><i>Brca2^Δ/Δ  = </i> deletion mutant for <i>Brca2</i> gene.</p><p><i>p53^Δ/Δ</i>  =  deletion mutant for p53 gene.</p><p><i>p53^R172H/Δ</i>  = point mutation and deletion mutant for <i>p53</i> gene.</p

Assessment of histopathological changes induced by drug treatment in <i>Brca1</i>-wild type and -deficient tumor lines.

<p>A, An example of histology and IHC of <i>TgK18G_T121^tg/+/Brca1^Δ/Δ/p53^Δ/Δ</i> tumors treated with cisplatin and/or olaparib (a–l). Tumor line 39877 was sensitive to drug treatment, which resulted in decreased proliferation (Ki67) (A e–h) and increased DNA damage (γ-H2AX) (A i–l). Olaparib treatment resulted in increased nuclear size and pleiomorphism (A, b, f, j) as well as decreased degree of papillary differentiation. Note the marked increase in tumor multinucleated giant cells in the cisplatin (A, c, g, k) and combination treated (A, d, h, l). Scale bar represents 100 µm. Quantitative analysis of Ki67 (B), and γH2AX (C) in 3 different tumor lines. Proliferation rate is expressed as the percentage of Ki67 positive nuclei (brown-DAB) to the total number of nuclei in the tumor section (brown-DAB + blue-hematoxylin counter-stained negative nuclei). γH2AX is expressed as the percentage of total nuclear area (blue-hematoxylin counter-stain) to the total area positive for γH2AX (brown-DAB). Statistical differences between groups were analyzed by one-way ANOVA and Tukey's multiple comparisons test. Each point represents one animal. V; vehicle, O; olaparib, C; cisplatin, O+C; olaparib and cisplatin.</p

Effect of long term treatment on mouse survival.

<p>A, RTV measurements in survival study comparing the effect of different treatments on tumor development. RTV compares tumor volume at any given time point to the baseline (pre-dosing) tumor volume. Each point represents an individual animal. B, Kaplan-Mayer graph of mouse survival after long term treatment. Dashed line represents cessation of treatment at Day 68. Three out of 12 mice treated with combination therapy died early in the study and although they presented with small ovarian tumors they did not succumb to metastatic SEOC. Histopathological signs of mild nephrosis as a result of cytotoxicity have been observed in their tissues [V; vehicle (n = 10), O; olaparib (n = 12), C; cisplatin (n = 10), O+C; olaparib and cisplatin (n = 12)]. C, effect of second round of platinum or combination treatment following the tumor relapses in mice. Tumor volumes were determined by US imaging. Dashed line represents end of first round of treatment, dotted lines represent start of the second round of treatment (two different start dates for 2 different mouse groups).</p

Development and characterization of orthotopic models for SEOC.

<p>A, Allograft models of SEOC were generated by transplantation of an ovarian tumor fragments from the <i>de novo</i> models of SEOC under the bursae of syngeneic immunocompetent mice. Primary ovarian carcinoma cell lines were generated simultaneously. The latency for tumor development in orthotopic models shortened substantially compared to the latency of the <i>de novo</i> model. B, H&E of primary tumors (PT) and corresponding passage 1 (p1) tumors from 4 different tumor lines indicating SEOC histology in PT and derived orthotopic tumor transplants. Scale bar represents 100 µm. C, Principal component analysis of normal ovarian surface epithelium, primary ovarian tumors and different passages of derived orthotopic tumors. D, Cluster analysis of merged human and mouse data using classifier gene sets showed that passaged tumors, similarly to primary tumors, represented all 4 subgroups of human SEOC originally derived from TCGA study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095649#pone.0095649-CancerGenomeAtlasResearch1" target="_blank">[5]</a>.</p