Robert T. Kitchens

Endocrine-Therapy-Resistant ESR1 Variants Revealed by Genomic Characterization of Breast-Cancer-Derived Xenografts

To characterize patient-derived xenografts (PDXs) for functional studies, we made whole-genome comparisons with originating breast cancers representative of the major intrinsic subtypes. Structural and copy number aberrations were found to be retained with high fidelity. However, at the single-nucleotide level, variable numbers of PDX-specific somatic events were documented, although they were only rarely functionally significant. Variant allele frequencies were often preserved in the PDXs, demonstrating that clonal representation can be transplantable. Estrogen-receptor-positive PDXs were associated with ESR1 ligand-binding-domain mutations, gene amplification, or an ESR1/YAP1 translocation. These events produced different endocrine-therapy-response phenotypes in human, cell line, and PDX endocrine-response studies. Hence, deeply sequenced PDX models are an important resource for the search for genome-forward treatment options and capture endocrine-drug-resistance etiologies that are not observed in standard cell lines. The originating tumor genome provides a benchmark for assessing genetic drift and clonal representation after transplantation.

Genetic heterogeneity of lipoproteins in inbred strains of mice: Analysis by gel-permeation chromatography☆

To assess genetic variation of murine lipoprotein profiles, plasma lipoproteins of 11 inbred strains, AKR/J, BALB/cByJ, C3H/HeJ, C57BL/6J, C57BL/6ByJ, C57L/J, DBA/1LacJ, 129/J, NZB/B1NJ, PL/J, and SWR/J, were analyzed by gel-permeation chromatography (fast peptide liquid chromatography) and nondenaturing gradient gel electrophoresis. Vena caval blood was drawn after 18 to 20 hours of fasting. Plasma triglyceride and cholesterol concentrations ranged from 12.9 mg/dL (C57BL/6ByJ) to 66.9 mg/dL (C3H/HeJ) and from 54.8 mg/dL (AKR/J) to 128.5 mg/dL (NZB/B1NJ), respectively. Mouse strain-related heterogeneities of very low-, low-, and high-density lipoprotein (VLDL, LDL, and HDL, respectively) concentrations were documented; VLDL-triglyceride concentrations ranged from 7.5 mg/dL to 38.8 mg/dL, LDL cholesterol from 12.0 mg/dL to 39.6 mg/dL, and HDL cholesterol from 41.3 mg/dL to 92.4 mg/dL. Hyper-VLDL-triglyceridemia was present in C3H/HeJ and SWR/J strains and hyper-LDL-cholesterolemia in NZB/B1NJ, C3H/HeJ, and DBA/1LacJ. VLDL cholesterol/VLDL triglyceride ratios also ranged widely among strains (0.13 to 0.43), with C57BL/6J, C57BL/6ByJ, and C57L/J, the strains particularly susceptible to diet-induced atherosclerosis, having the highest VLDL-lipid ratio. LDL and HDL size heterogeneities were also observed. LDL and HDL diameters ranged between 24.1 nm and 29.4 nm, and between 9.24 nm and 10.32 nm, respectively. Although LDL sizes showed no segregation, HDL sizes fell into two groups. C57L/J and C57BL/6J possessed low HDL-cholesterol concentrations and small-sized HDL. HDL sizes were positively correlated with HDL-cholesterol concentrations (r = .90, P less than .001) and LDL-cholesterol concentrations (r = .85, P less than .001), but LDL sizes did not correlate with lipoprotein concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Controls for Lung Dendritic Cell Maturation and Migration during Respiratory Viral Infection

Dendritic cells are ideally suited to orchestrate the innate and adaptive immune responses to infection, but we know little about how these cells respond to infection with common respiratory viruses. Paramyxoviral infections are the most frequent cause of serious respiratory illness in childhood and are associated with an increased risk of asthma. We therefore used a high-fidelity mouse model of paramyxoviral respiratory infection triggered by Sendai virus to examine the response of conventional and plasmacytoid dendritic cells (cDCs and pDCs, respectively) in the lung. We found that pDCs are scarce at baseline but become the predominant population of lung dendritic cells during infection. This recruitment allows for a source of IFN-α locally at the site of infection. In contrast, cDCs rapidly differentiate into myeloid cDCs and begin to migrate from the lung to draining lymph nodes within 2 h after viral inoculation. These events cause the number of lung cDCs to decrease rapidly and remain decreased at the site of viral infection. Maturation and migration of lung cDCs depends on Ccl5 and Ccr5 signals because these events are significantly impaired in Ccl5−/− and Ccr5−/− mice. cDCs failure to migrate to draining lymph nodes in Ccl5−/− or Ccr5−/− mice is associated with impaired up-regulation of CCR7 that would normally direct this process. Our results indicate that pDCs and cDCs respond distinctly to respiratory paramyxoviral infection with patterns of movement that should serve to coordinate the innate and adaptive immune responses, respectively.

In vivo regulation of low-density lipoprotein receptor and apolipoprotein B gene expressions by dietary fat and cholesterol in inbred strains of mice

Two proteins that may be important in the hypercholesterolemia and atherosclerosis produced by dietary fat and/or cholesterol are apoB and the LDL-receptor. We evaluated the molecular and genetic regulation of these two proteins by two important components of atherogenic diets: dietary fatty acids and dietary cholesterol. The control diet (C) contained 5% corn oil; the high cholesterol (HC) diets, 5% corn oil plus 0.5% or 2% cholesterol; the high fat diet (HF) 1% corn oil and 20% hydrogenated coconut oil; the fat plus cholesterol diets (HF/C) were the same as HF diet plus either 0.5% or 2% cholesterol. Ten strains of inbred mice were fed the C and HF/C (2% cholesterol) diets. Three strains; C3H, C57BL and SWR, were studied in greater detail. In them the effects of dietary fat and cholesterol were assessed separately and together. These three strains were fed all six diets. Lipoprotein profiles of plasma and indexes of lipoprotein composition were obtained by gel filtration chromatography and in selected strains by gradient ultracentrifugation. Relative rates of transcription of LDL-receptor mRNA and apoB mRNA were measured in purified mouse liver nuclei and levels of LDL-receptor mRNA and apoB mRNA in liver and intestine were quantified by RNA excess solution hybridization assays. The HF/C diet produced rises in plasma total-, VLDL- and LDL-cholesterol and apoB concentrations in the ten strains. VLDL and LDL became cholesterol-enriched and the proportion of total cholesterol transported in VLDL and LDL rose at the expense of HDL. This general pattern of HF/C diet-induced changes was similar in all strains, but there were marked quantitative differences between strains with respect to lipid and lipoprotein concentrations, and compositions and the distribution of cholesterol on both the HC and HF/C diets. The strain-related differences were not due to differences in absorption of dietary cholesterol because, for any given diet, hepatic cholesterol levels increased to the same extent in all strains. Nor were the strain-related differences related to alleles of the apoB gene as determined by RFLP analyses. In the three strains, hepatic LDL-receptor mRNA transcription was suppressed by all diets. But, LDL-receptor mRNA levels in both intestine and liver were suppressed only by the HC and HF/C diets and not by the HF diet. Thus, dietary cholesterol decreased LDL-receptor mRNA levels by mechanisms operating at the transcriptional level, while dietary fatty acids, in addition to inhibiting transcription also appeared to enhance mRNA stability.(ABSTRACT TRUNCATED AT 400 WORDS)

Dietary fatty acids and dietary cholesterol differ in their effect on the in vivo regulation of apolipoprotein A-I and A-II gene expression in inbred strains of mice

Dietary cholesterol and dietary saturated fatty acids affected the plasma concentrations of various HDL components and the hepatic and intestinal expression of the apolipoprotein (apo) A-I gene and the hepatic expression of the A-II gene differently in three inbred strains of female mice. Thus, the HC diet (0.5% cholesterol, no added fatty acids) decreased HDL-cholesterol in C57BL and SWR strains but not in the C3H strain; plasma apo A-I and apo A-II concentrations decreased in all three strains. HDL-C/apo A-I and apo A-I/apo A-II mass ratios increased, suggesting that the HC diet altered both the concentrations and the compositions of HDL particles. In contrast, the HF diet (20% hydrogenated coconut oil, no added cholesterol) increased HDL cholesterol and apo A-I concentrations. The combination diet (HF/C, 20% coconut oil plus 0.5% cholesterol) increased HDL cholesterol and decreased triacylglycerols. Apo A-I concentrations were unaltered except for a significant increase in SWR mice. Apo A-II concentrations decreased in all strains. To examine molecular events that could lead to the changes in plasma apo A-I and apo A-II, we measured transcription rates in hepatic nuclei and steady state mRNA concentrations in liver and intestine and apo A-I synthetic rates in liver. Dietary cholesterol and fatty acids produced differing effects at transcriptional as well as post-transcriptional loci and the changes differed according to mouse strain. The most pronounced strain-related differences for both apo A-I and apo A-II occurred at post-transcriptional loci of apoprotein production. These could represent altered rates of translation in, or secretion from liver and/or intestine, or altered rates of clearance from plasma. In conclusion, the regulation of apo A-I and apo A-II gene expression by diet occurs at several steps of their production and perhaps also in catabolic pathways. This study identifies potential loci of regulation and forms the basis for future studies investigating specific genetic and molecular regulatory mechanisms.

Cutting Edge: CD49d+ Neutrophils Induce FcεRI Expression on Lung Dendritic Cells in a Mouse Model of Postviral Asthma

The increasing prevalence of atopy and asthma remains unexplained but may be due to infection with respiratory viruses. In support of this hypothesis, we showed that experimental asthma after viral infection in mice depended on type I IFN-driven upregulation of FcεRI on conventional dendritic cells (cDCs) in the lung. In this article, we demonstrate that FcεRI expression on lung cDCs depends on an unexpected activity of a CD49d+ subset of polymorphonuclear neutrophils (PMNs) that are found in the lungs of wild-type C57BL6 but not mice deficient in type I IFNR. Expression of FcεRI depends in part on a CD11b-dependent interaction between PMNs and cDCs. This study demonstrates a PMN–cDC interaction in the lung that is necessary for the ability of viral infection to induce atopic disease.

Cutting edge: CD49d+ neutrophils induce FcepsilonRI expression on lung dendritic cells in a mouse model of postviral asthma.

The increasing prevalence of atopy and asthma remains unexplained but may be due to infection with respiratory viruses. In support of this hypothesis, we showed that experimental asthma after viral infection in mice depended on type I IFN-driven upregulation of FcεRI on conventional dendritic cells (cDCs) in the lung. In this article, we demonstrate that FcεRI expression on lung cDCs depends on an unexpected activity of a CD49d+ subset of polymorphonuclear neutrophils (PMNs) that are found in the lungs of wild-type C57BL6 but not mice deficient in type I IFNR. Expression of FcεRI depends in part on a CD11b-dependent interaction between PMNs and cDCs. This study demonstrates a PMN–cDC interaction in the lung that is necessary for the ability of viral infection to induce atopic disease.

Abstract 5544: Patient-derived xenograft study reveals the pharmacology and the role of ESR1 gene aberrations in endocrine therapy resistance of ER positive breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: The study of estrogen-receptor-positive patient-derived xenografts (PDXs) from patients with disease progression despite endocrine therapy has revealed recurrent ESR1 mutation, gene amplification and ESR1 gene translocations as likely drivers of resistance. We addressed this hypothesis further using pharmacological and genetic approaches in the PDX system. Methodology: ER positive PDXs that harbored one of four recurrent ligand binding domain (LBD) mutations (Y537S/N/C and D538G) as well as a ESR1/YAP1 translocation were studied in parallel with T47D and MCF7 cells expressing these mutants through lentivirus constructs. Single cell suspensions generated from PDX models were using for in vitro experiments using with a panel of endocrine inhibitors and ESR1 shRNA constructs. In vitro effects were confirmed using in vivo tumor response. Results: Cell suspensions from ER+ PDX models in short-term culture successfully reproduced the endocrine therapy resistant phenotypes exhibited by the donor patient, thereby facilitating drugs screens designed to identify new endocrine approaches for ESR1 mutant breast cancer using authentic models rather than an exclusive reliance on models derived by transfection of mutant constructs into standard indicator cell lines. We were also able to demonstrate that that lentivirus-driven shRNA mediated knockdown of ESR1-YAP1 caused inhibition of PDX tumor growth in in vivo and reduced cell growth in vitro, confirming that this chimeric protein is a driver of estradiol-independent growth. Conclusion: ER+ PDX can be manipulated in vitro and in vivo to successfully model ESR1 mutation and gene-rearrangement driven endocrine therapy resistance. PDX studies complement standard ectopic expression systems for the study of ESR1 mutation-driven resistance and, we hypothesize, will provide pharmacological data that will predict the activity of pharmacological agents designed to improve outcomes for patients with ESR1 mutant driven endocrine therapy resistance. Citation Format: Triparna Sen, Shunqiang Li, Jieya Shao, Robert Crowder, Robert Kitchens, Matthew J. Ellis. Patient-derived xenograft study reveals the pharmacology and the role of ESR1 gene aberrations in endocrine therapy resistance of ER positive breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5544. doi:10.1158/1538-7445.AM2014-5544

CD49d+ neutrophils induce FcεRI expression on lung dendritic cells in a mouse model of postviral asthma

The increasing prevalence of atopy and asthma remains unexplained but may be due to infection with respiratory viruses. In support of this hypothesis, we showed that experimental asthma after viral infection in mice depended on type I IFN-driven upregulation of FcεRI on conventional dendritic cells (cDCs) in the lung. In this article, we demonstrate that FcεRI expression on lung cDCs depends on an unexpected activity of a CD49d+ subset of polymorphonuclear neutrophils (PMNs) that are found in the lungs of wild-type C57BL6 but not mice deficient in type I IFNR. Expression of FcεRI depends in part on a CD11b-dependent interaction between PMNs and cDCs. This study demonstrates a PMN–cDC interaction in the lung that is necessary for the ability of viral infection to induce atopic disease.

Induction of high-affinity IgE receptor on lung dendritic cells during viral infection leads to mucous cell metaplasia

Grayson et al. 2007. J. Exp. Med. doi:10.1084/jem.20070360 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1084%252Fjem.20070360%26rft_id%253Dinfo%253Apmid%252F17954569%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%