Amanda Koehne

The Influence of Shc Proteins on Life Span in Mice

The signaling molecule p66Shc is often described as a longevity protein. This conclusion is based on a single life span study that used a small number of mice. The purpose of the present studies was to measure life span in a sufficient number of mice to determine if longevity is altered in mice with decreased Shc levels (ShcKO). Studies were completed at UC Davis and the European Institute of Oncology (EIO). At UC Davis, male C57BL/6J WT and ShcKO mice were fed 5% or 40% calorie-restricted (CR) diets. In the 5% CR group, there was no difference in survival curves between genotypes. There was also no difference between genotypes in prevalence of neoplasms or other measures of end-of-life pathology. At 40% calorie restriction group, 70th percentile survival was increased in ShcKO, while there were no differences between genotypes in median or subsequent life span measures. At EIO, there was no increase in life span in ShcKO male or female mice on C57BL/6J, 129Sv, or hybrid C57BL/6J-129Sv backgrounds. These studies indicate that p66Shc is not a longevity protein. However, additional studies are needed to determine the extent to which Shc proteins may influence the onset and severity of specific age-related diseases.

To the Root of the Curl: A Signature of a Recent Selective Sweep Identifies a Mutation That Defines the Cornish Rex Cat Breed

The cat (Felis silvestris catus) shows significant variation in pelage, morphological, and behavioral phenotypes amongst its over 40 domesticated breeds. The majority of the breed specific phenotypic presentations originated through artificial selection, especially on desired novel phenotypic characteristics that arose only a few hundred years ago. Variations in coat texture and color of hair often delineate breeds amongst domestic animals. Although the genetic basis of several feline coat colors and hair lengths are characterized, less is known about the genes influencing variation in coat growth and texture, especially rexoid – curly coated types. Cornish Rex is a cat breed defined by a fixed recessive curly coat trait. Genome-wide analyses for selection (di, Tajima’s D and nucleotide diversity) were performed in the Cornish Rex breed and in 11 phenotypically diverse breeds and two random bred populations. Approximately 63K SNPs were used in the analysis that aimed to localize the locus controlling the rexoid hair texture. A region with a strong signature of recent selective sweep was identified in the Cornish Rex breed on chromosome A1, as well as a consensus block of homozygosity that spans approximately 3 Mb. Inspection of the region for candidate genes led to the identification of the lysophosphatidic acid receptor 6 (LPAR6). A 4 bp deletion in exon 5, c.250_253_delTTTG, which induces a premature stop codon in the receptor, was identified via Sanger sequencing. The mutation is fixed in Cornish Rex, absent in all straight haired cats analyzed, and is also segregating in the German Rex breed. LPAR6 encodes a G protein-coupled receptor essential for maintaining the structural integrity of the hair shaft; and has mutations resulting in a wooly hair phenotype in humans.

The Influence of Dietary Fat Source on Life Span in Calorie Restricted Mice

Calorie restriction (CR) without malnutrition extends life span in several animal models. It has been proposed that a decrease in the amount of polyunsaturated fatty acids (PUFAs), and especially n-3 fatty acids, in membrane phospholipids may contribute to life span extension with CR. Phospholipid PUFAs are sensitive to dietary fatty acid composition, and thus, the purpose of this study was to determine the influence of dietary lipids on life span in CR mice. C57BL/6J mice were assigned to four groups (a 5% CR control group and three 40% CR groups) and fed diets with soybean oil (high in n-6 PUFAs), fish oil (high in n-3 PUFAs), or lard (high in saturated and monounsaturated fatty acids) as the primary lipid source. Life span was increased (p < .05) in all CR groups compared to the Control mice. Life span was also increased (p < .05) in the CR lard mice compared to animals consuming either the CR fish or soybean oil diets. These results indicate that dietary lipid composition can influence life span in mice on CR, and suggest that a diet containing a low proportion of PUFAs and high proportion of monounsaturated and saturated fats may maximize life span in animals maintained on CR.

Japanese Bobtail: vertebral morphology and genetic characterization of an established cat breed

Several cat breeds are defined by morphological variation of the tail. The Japanese Bobtail is a breed that has been accepted for registration only within the past 50 years; however, the congenital kinked tail variants defining this breed were documented in the Far East centuries ago and the cats are considered ‘good luck’ in several Asian cultures. The recent discovery of the mutation for the tailless Manx phenotype has demonstrated that the Japanese Bobtail does not have a causative mutation in the same gene (T-Box). Here, a simple segregation analysis of cats bred from a pedigreed Japanese Bobtail demonstrated a simple autosomal dominant mode of inheritance with variable expression of the tail length and kink placement. Unexpectedly, radiological examinations of the entire vertebral column of kink-tailed cats indicated variation from the normal vertebral feline formula (C7, T13, L7, S3, Cd20–24), including cats with mostly one reduction of thoracic vertebrae (C7, T12, L7, S3), and an average of 15.8 caudal vertebrae. A few cats had variation in the number of cervical vertebrae. Several transitional vertebrae and anomalous ribs were noted. One cat had a bifid vertebra in the tail. Most cats had hemivertebrae that were usually included in the tail kink, one of which was demonstrated by gross pathology and histopathology. The abnormal vertebral formula or the placement of the kink in the tail did not coincide with morbidity or mortality.

Mutant mice derived by ICSI of evaporatively dried spermatozoa exhibit expected phenotype

Apolipoprotein E (Apoe)-deficient knockout mice were used to test the hypothesis that mutant mice preserved as evaporatively dried (ED) spermatozoa, stored at -80 °C for 6 months, and then recovered by ICSI will exhibit the same phenotype as before preservation. The birth rate of mice recovered by ICSI of evaporatively dried spermatozoa was lower than that of fresh spermatozoa (17.5 vs 38.0%). Progeny of mice preserved using evaporatively dried spermatozoa were reproductively sound. From these, the second generation of mice produced by natural mating showed lesions typical of APOE deficiency, including severe hypercholesterolemia, hypertriglyceridemia, markedly increased plasma low-density lipoprotein level, and extensive and severe atherosclerotic lesions in the aorta. We conclude that the expected phenotype caused by an induced genetic mutation can be faithfully recapitulated and sustained in subsequent generations of mice preserved and stored as ED spermatozoa and recovered using ICSI. Because it is simpler, faster, and cheaper than conventional (cryopreservation) and nonconventional (freeze-drying) preservation procedures, evaporative drying is a viable, cost-effective, and efficient method for preserving and storing valuable mutant mouse strains.

Mandibular odontoameloblastoma in a rat and a horse

Odontoameloblastoma (OA) is a mixed odontogenic tumor that is an ameloblastoma with concurrent histologic evidence of odontoma differentiation. As a mixed tumor, OA is a tripartite lesion comprised of neoplastic odontogenic epithelium, induced dental ectomesenchyme (dental pulp), and mineralized dental matrix. Although rare, OA represents a diagnostic conundrum, as it is histologically closely related to 2 other mixed odontogenic tumors: odontoma (complex and compound) and ameloblastic fibro-odontoma. Herein we describe an OA arising from the mandible of a 4-mo-old Fischer 344 rat that had been exposed in utero to the mutagen ENU (N-ethyl-N-nitrosourea), and a naturally occurring lesion in a 2-y-old Appaloosa horse. In order to satisfy the diagnostic criteria for this lesion, mineralized dental matrix in relationship to neoplastic odontogenic epithelium must be identifiable within the OA lesion. This group of odontogenic tumors is differentiated by the degree to which the dental matrix is organized and the relative proportions of pulp ectomesenchyme, odontogenic matrix, and odontogenic epithelium.

Glucose Uptake and Intracellular pH in a Mouse Model of Ductal Carcinoma In situ (DCIS) Suggests Metabolic Heterogeneity

Mechanisms for the progression of ductal carcinoma in situ (DCIS) to invasive breast carcinoma remain unclear. Previously we showed that the transition to invasiveness in the mammary intraepithelial neoplastic outgrowth (MINO) model of DCIS does not correlate with its serial acquisition of genetic mutations. We hypothesized instead that progression to invasiveness depends on a change in the microenvironment and that precancer cells might create a more tumor-permissive microenvironment secondary to changes in glucose uptake and metabolism. Immunostaining for glucose transporter 1 (GLUT1) and the hypoxia marker carbonic anhydrase 9 (CAIX) in tumor, normal mammary gland and MINO (precancer) tissue showed differences in expression. The uptake of the fluorescent glucose analog dye, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG), reflected differences in the cellular distributions of glucose uptake in normal mammary epithelial cells (nMEC), MINO, and Met1 cancer cells, with a broad distribution in the MINO population. The intracellular pH (pHi) measured using the fluorescent ratio dye 2′,7′-bis(2-carboxyethyl)-5(6)-155 carboxyfluorescein (BCECF) revealed expected differences between normal and cancer cells (low and high, respectively), and a mixed distribution in the MINO cells, with a subset of cells in the MINO having an increased rate of acidification when proton efflux was inhibited. Invasive tumor cells had a more alkaline baseline pHi with high rates of proton production coupled with higher rates of proton export, compared with nMEC. MINO cells displayed considerable variation in baseline pHi that separated into two distinct populations: MINO high and MINO low. MINO high had a noticeably higher mean acidification rate compared with nMEC, but relatively high baseline pHi similar to tumor cells. MINO low cells also had an increased acidification rate compared with nMEC, but with a more acidic pHi similar to nMEC. These findings demonstrate that MINO is heterogeneous with respect to intracellular pH regulation which may be associated with an acidified regional microenvironment. A change in the pH of the microenvironment might contribute to a tumor-permissive or tumor-promoting progression. We are not aware of any previous work showing that a sub-population of cells in in situ precancer exhibits a higher than normal proton production and export rate.

Abstract A35: Characterization of the genomic landscape of osteosarcoma metastasis

Early metastasis to the lungs is a cardinal feature of osteosarcoma (OS), and complications from metastatic disease remain the most common cause of cancer-related death. Despite the prevalence of metastasis in OS, the pathogenesis is poorly understood. We have established a collection of 16 patient-derived xenografts (PDX) from human OS tumors, both from the primary site as well as metastasis. Our collection includes 9 diagnostic biopsies from primary site tumors, 2 primary tumor resections, 3 lung metastases, and 1 ascites fluid metastasis. Gene expression analysis of an initial set of 5 PDX models (3 metastases, 2 primaries) has been completed. A more extensive analysis of all 16 models by RNAseq and WGS is currently underway. Preliminary gene expression analysis identified a significant number of genes differentially expressed between metastatic samples and non-metastatic samples. This list included genes associated with cell adhesion and motility such as MEGF10, genes associated with endochondral ossification and bone remodeling such as RANKL, and genes associated with deposition of extracellular matrix (ECM) such as COL21A. Another candidate gene, ENPP1, is responsible for mineralization of the ECM and has been implicated in breast cancer metastasis to bone. We have validated differential expression of a number of these genes in independent samples of metastatic OS. We are currently using small hairpin RNA (shRNA), CRISPR, and overexpression vectors to knockdown, silence, and overexpress ENPP1 and other candidate genes in OS cell lines. Using in vitro migration and invasion assays as well as intravenous injection and orthotopic mouse models, we are characterizing the contribution of candidate genes to the metastatic propensity of OS. Citation Format: Amanda L. Koehne, Leanne C. Sayles, Marcus R. Breese, Dedeepya Vaka, Alejandro Sweet-Cordero. Characterization of the genomic landscape of osteosarcoma metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A35.

Addisonian Crisis due to Metastatic Adenocarcinoma in a Pygmy Goat

A 15-year-old Pygmy doe was evaluated for acute onset of lethargy, anorexia, and weakness. Adrenal insufficiency was diagnosed based on physical exam findings, blood work abnormalities (hyponatremia, hyperkalemia, azotemia, and hypoglycemia), and lack of cortisol response to the ACTH stimulation test. Abdominal ultrasound exam revealed an intact urinary tract and multiple bilateral peri-renal masses. The doe was treated with intravenous fluid therapy aimed at correcting the electrolyte abnormalities and intravenous corticosteroids. She responded favorably to medical therapy in 24 hours, with dramatic improvement in attitude and appetite. Fluid therapy was discontinued, and the doe was discharged from the hospital on steroid supplementation. She deteriorated rapidly and died at home 36 hours after discharge. Necropsy results revealed metastatic adenocarcinoma originating from the uterus that infiltrated the urinary bladder, the region of the adrenal glands, the left and right renal lymph nodes, the left kidney, the caudal vena cava, the submandibular lymph nodes, the diaphragm, the lungs, and the omentum. Addison’s syndrome in ruminants should be considered as an uncommon sequel of intra-abdominal neoplastic processes.