The prolactin receptor: A cross-species comparison of gene structure, transcriptional regulation, tissue-specificity, and genetic variation.

The conserved and multifaceted functions of prolactin (PRL) are coordinated through varied distribution and expression of its cell-surface receptor (PRLR) across a range of tissues and physiological states. The resultant heterogeneous expression of PRLR mRNA and protein across different organs and cell types supports a wide range of PRL-regulated processes including reproduction, lactation, development, and homeostasis. Genetic variation within the PRLR gene also accounts for several phenotypes impacting agricultural production and human pathology. The goal of this review is to highlight the many elements that control differential expression of the PRLR across tissues, and the various phenotypes that exist across species due to variation in the PRLR gene.

Transcriptomic changes underlying glucocorticoid-induced suppression of milk production by dairy cows.

Milk production by dairy cows is sensitive to increased levels of stress hormones such as glucocorticoids (GC) that also regulate the transcription of several genes required for milk synthesis. Whereas previous studies identified that an exogenous GC such as dexamethasone (DEX) transiently suppresses milk yield in several species without any pronounced effect on milk protein or fat percentage, the mechanism underlying this effect has not been established. In this study we sought to establish changes within the mammary glands of non-pregnant dairy cows in their second lactation (n = 3-4; 648-838 kg) following a single dose of exogenous DEX. Changes in the udder were monitored by serial biopsy of alternating quarters, concurrent with quarter-level monitoring of milk yield and composition. Dexamethasone increased serum glucose levels from 12-36 h (p <0 .05), reduced milk yield from 12-48 h (p <0 .05), increased % milk protein content at 24 h post-DEX, and transiently decreased both milk lactose and α-lactalbumin content, while not altering the level of milk fat. After 72 h, all aspects of milk production had returned to pre-treatment levels. Transcriptomic changes in the mammary glands in response to DEX were identified by RNA sequencing followed by differential gene expression analysis. Coincident with the milk yield and composition changes was the differential expression of 519 and 320 genes at 12 and 24 h after DEX (adjusted p <0 .05), respectively, with the return of all gene expression to baseline levels by 72 h. Among the transcriptomic changes in response to DEX, there was notable downregulation of elements in the lactose synthesis pathway, specifically AQP3, GALE and LALBA (α-lactalbumin) at 12 h, and sustained downregulation of LALBA at 24 h. One gene in the pathway, UGP2, was upregulated at 12-24 h post-DEX. This work supports the hypothesis that there is a direct relationship between the response to DEX and the concurrent suppression of milk yield due to the reduced synthesis of α-lactalbumin and lactose by the mammary epithelium. The ability of glucocorticoids to modulate the homeorrhetic requirements for glucose during stressful states concurrent with immune activation bears significance for dairy animals as well as a broad range of lactating mammals.

LincRNA#1 knockout alone does not affect polled phenotype in cattle heterozygous for the celtic POLLED allele.

A long intergenic non-coding RNA (lincRNA#1) is overexpressed in the horn bud region of polled (hornless) bovine fetuses, suggesting a potential role in horn bud suppression. Genome editing was used to test whether the absence of this sequence was associated with the horned phenotype. Two gRNAs with high mutation efficiencies targeting the 5' and the 3' regions flanking the lincRNA#1 sequence were co-injected with Cas9 as ribonucleoprotein complexes into bovine zygotes (n = 121) 6 h post insemination. Of the resulting blastocysts (n = 31), 84% had the expected 3.7 kb deletion; of these embryos with the 3.7 kb deletions, 88% were biallelic knockouts. Thirty-nine presumptive edited 7-day blastocysts were transferred to 13 synchronized recipient cows resulting in ten pregnancies, five with embryos heterozygous for the dominant PC POLLED allele at the POLLED locus, and five with the recessive pp genotype. Eight (80%) of the resulting fetuses were biallelic lincRNA#1 knockouts, with the remaining two being mosaic. RT-qPCR analysis was used to confirm the absence of lincRNA#1 expression in knockout fetuses. Phenotypic and histological analysis of the genotypically (PCp) POLLED, lincRNA#1 knockout fetuses revealed similar morphology to non-edited, control polled fetuses, indicating the absence of lincRNA#1 alone does not result in a horned phenotype.

Unique Transcriptomic Changes Underlie Hormonal Interactions During Mammary Histomorphogenesis in Female Pigs.

Successful lactation and the risk for developing breast cancer depend on growth and differentiation of the mammary gland (MG) epithelium that is regulated by ovarian steroids (17ß-estradiol [E] and progesterone [P]) and pituitary-derived prolactin (PRL). Given that the MG of pigs share histomorphogenic features present in the normal human breast, we sought to define the transcriptional responses within the MG of pigs following exposure to all combinations of these hormones. Hormone-ablated female pigs were administered combinations of E, medroxyprogesterone 17-acetate (source of P), and either haloperidol (to induce PRL) or 2-bromo-α-ergocryptine. We subsequently monitored phenotypic changes in the MG including mitosis, receptors for E and P (ESR1 and PGR), level of phosphorylated STAT5 (pSTAT5), and the frequency of terminal ductal lobular unit (TDLU) subtypes; these changes were then associated with all transcriptomic changes. Estrogen altered the expression of approximately 20% of all genes that were mostly associated with mitosis, whereas PRL stimulated elements of fatty acid metabolism and an inflammatory response. Several outcomes, including increased pSTAT5, highlighted the ability of E to enhance PRL action. Regression of transcriptomic changes against several MG phenotypes revealed 1669 genes correlated with proliferation, among which 29 were E inducible. Additional gene expression signatures were associated with TDLU formation and the frequency of ESR1 or PGR. These data provide a link between the hormone-regulated genome and phenome of the MG in a species having a complex histoarchitecture like that in the human breast, and highlight an underexplored synergy between the actions of E and PRL during MG development.

In Utero Exposure to trans-10, cis-12 Conjugated Linoleic Acid Modifies Postnatal Development of the Mammary Gland and its Hormone Responsiveness.

We previously showed that dietary trans-10, cis-12 conjugated linoleic acid (10,12 CLA) stimulates estrogen-independent mammary growth in young ovariectomized mice. Here we investigated the effects of in utero or postnatal exposure to cis-9, trans-11 (9,11 CLA) and 10,12 CLA on postnatal development of the mammary gland and its responsiveness to ovarian steroids. In the first experiment we fed dams different CLA prior to and during gestation, then cross fostered female pups onto control fed dams prior to assessing the histomorphology of their mammary glands. Pregnant dams in the second experiment were similarly exposed to CLA, after which their female pups were ovariectomized then treated with 17ß-estradiol (E), progesterone (P) or E + P for 5 days. In a third experiment, mature female mice were fed different CLA for 28 days prior to ovariectomy, then treated with E, P or E + P. Our data indicate that 10,12 CLA modifies the responsiveness of the mammary glands to E or E + P when exposure occurs either in utero, or postnatally. These findings underline the sensitivity of the mammary glands to dietary fatty acids and reinforce the potential for maternal nutrition to impact postnatal development of the mammary glands and their risk for developing cancer.

One-step generation of a targeted knock-in calf using the CRISPR-Cas9 system in bovine zygotes.

BACKGROUND: The homologous recombination (HR) pathway is largely inactive in early embryos prior to the first cell division, making it difficult to achieve targeted gene knock-ins. The homology-mediated end joining (HMEJ)-based strategy has been shown to increase knock-in efficiency relative to HR, non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ) strategies in non-dividing cells. RESULTS: By introducing gRNA/Cas9 ribonucleoprotein complex and a HMEJ-based donor template with 1 kb homology arms flanked by the H11 safe harbor locus gRNA target site, knock-in rates of 40% of a 5.1 kb bovine sex-determining region Y (SRY)-green fluorescent protein (GFP) template were achieved in Bos taurus zygotes. Embryos that developed to the blastocyst stage were screened for GFP, and nine were transferred to recipient cows resulting in a live phenotypically normal bull calf. Genomic analyses revealed no wildtype sequence at the H11 target site, but rather a 26 bp insertion allele, and a complex 38 kb knock-in allele with seven copies of the SRY-GFP template and a single copy of the donor plasmid backbone. An additional minor 18 kb allele was detected that looks to be a derivative of the 38 kb allele resulting from the deletion of an inverted repeat of four copies of the SRY-GFP template. CONCLUSION: The allelic heterogeneity in this biallelic knock-in calf appears to have resulted from a combination of homology directed repair, homology independent targeted insertion by blunt-end ligation, NHEJ, and rearrangement following editing of the gRNA target site in the donor template. This study illustrates the potential to produce targeted gene knock-in animals by direct cytoplasmic injection of bovine embryos with gRNA/Cas9, although further optimization is required to ensure a precise single-copy gene integration event.

Genetic Engineering of Livestock: The Opportunity Cost of Regulatory Delay.

Genetically engineered (GE) livestock were first reported in 1985, and yet only a single GE food animal, the fast-growing AquAdvantage salmon, has been commercialized. There are myriad interconnected reasons for the slow progress in this once-promising field, including technical issues, the structure of livestock industries, lack of public research funding and investment, regulatory obstacles, and concern about public opinion. This review focuses on GE livestock that have been produced and documents the difficulties that researchers and developers have encountered en route. Additionally, the costs associated with delayed commercialization of GE livestock were modeled using three case studies: GE mastitis-resistant dairy cattle, genome-edited porcine reproductive and respiratory syndrome virus-resistant pigs, and the AquAdvantage salmon. Delays of 5 or 10 years in the commercialization of GE livestock beyond the normative 10-year GE product evaluation period were associated with billions of dollars in opportunity costs and reduced global food security.

Folate Deficiency Inhibits Development of the Mammary Gland and its Associated Lymphatics in FVB Mice.

BACKGROUND: Folate is essential for DNA synthesis, DNA repair, cell proliferation, development, and morphogenesis. Folic acid (FA) is a nutritional supplement used to fortify human diets. OBJECTIVES: We investigated the effects of dietary FA on early mammary gland (MG) development and hyperplasia. METHODS: Study 1: nulliparous female FVB wild-type (WT) mice were fed control (Con; 2 mg FA/kg), deficient (Def; 0 mg FA/kg), excess (Ex; 5 mg FA/kg), or super excess (S-Ex; 20 mg FA/kg) diets for 8 wk before mating to WT or heterozygous FVB/N-Tg[mouse mammary tumor virus long terminal repeat (MMTV)-polyomavirus middle T antigen (PyVT)]634Mul/J (MMTV-PyMT+/-) transgenic males. Dams were fed these diets until they weaned WT or MMTV-PyMT+/- pups, which were fed the dam's diet from postnatal day (PND) 21 to 42. Tissues were collected from female progeny at PNDs 1, 21, and 42. Study 2: Con or Def diets were fed to WT intact females and males from PND 21 to 56, or to ovariectomized females from PND 21 to 77; tissues were collected at PND 56 or 77. Growth of all offspring, development of MGs, MG hyperplasia, supramammary lymph nodes, thymus and spleen, cell proliferation, and expression of MG growth factors were measured. RESULTS: Study 1: Ex or S-Ex did not affect postnatal MG development or hyperplasia. The rate of isometric MG growth (PND 1-21) was reduced by 69% in Def female progeny (P < 0.0001). Similarly, hyperplastic growth in MGs of Def MMTV-PyMT+/- offspring was 18% of Con (P < 0.05). The Def diet reduced supramammary lymph node size by 20% (P < 0.0001) and increased MG insulin-like growth factor 2 mRNA by 200% (P < 0.05) and protein by 130%-150% (P < 0.05). Study 2: the Def diet did not affect MG growth, but it did reduce supramammary lymph node size (P < 0.05), spleen weight (P < 0.001), and thymic medulla area (P < 0.05). CONCLUSIONS: In utero and postnatal folate deficiency reduced the isometric development of the MGs and early MG hyperplasia. Postnatal folate deficiency reduced the development of lymphatic tissues.

A high-throughput screening platform for Polycystic Kidney Disease (PKD) drug repurposing utilizing murine and human ADPKD cells.

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited monogenic disorders, characterized by a progressive decline in kidney function due in part to the formation of fluid-filled cysts. While there is one FDA-approved therapy, it is associated with potential adverse effects, and all other clinical interventions are largely supportive. Insights into the cellular pathways underlying ADPKD have revealed striking similarities to cancer. Moreover, several drugs originally developed for cancer have shown to ameliorate cyst formation and disease progression in animal models of ADPKD. These observations prompted us to develop a high-throughput screening platform of cancer drugs in a quest to repurpose them for ADPKD. We screened ~8,000 compounds, including compounds with oncological annotations, as well as FDA-approved drugs, and identified 155 that reduced the viability of Pkd1-null mouse kidney cells with minimal effects on wild-type cells. We found that 109 of these compounds also reduced in vitro cyst growth of Pkd1-null cells cultured in a 3D matrix. Moreover, the result of the cyst assay identified therapeutically relevant compounds, including agents that interfere with tubulin dynamics and reduced cyst growth without affecting cell viability. Because it is known that several ADPKD therapies with promising outcomes in animal models failed to be translated to human disease, our platform also incorporated the evaluation of compounds in a panel of primary ADPKD and normal human kidney (NHK) epithelial cells. Although we observed differences in compound response amongst ADPKD and NHK cell preparation, we identified 18 compounds that preferentially affected the viability of most ADPKD cells with minimal effects on NHK cells. Our study identifies attractive candidates for future efficacy studies in advanced pre-clinical models of ADPKD.

Author Correction: Genomic and phenotypic analyses of six offspring of a genome-edited hornless bull.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Anti-Cancer Activity of PAK4/NAMPT Inhibitor and Programmed Cell Death Protein-1 Antibody in Kidney Cancer.

BACKGROUND: Kidney cancer (or renal cell carcinoma, RCC) is the sixth most common malignancy in the United States and is increasing in incidence. Despite new therapies, including targeted therapies and immunotherapies, most RCCs are resistant to treatment. Thus, several laboratories have been evaluating new approaches to therapy, both with single agents as well as combinations. Although we have previously shown efficacy of the dual PAK4/nicotinamide phosphoribosyltransferase (NAMPT) inhibitor KPT-9274, and the immune checkpoint inhibitors (CPI) have shown utility in the clinic, there has been no evaluation of this combination either clinically or in an immunocompetent animal model of kidney cancer. METHODS: In this study, we use the renal cell adenocarcinoma (RENCA) model of spontaneous murine kidney cancer. Male BALB/cJ mice were injected subcutaneously with RENCA cells and, after tumors were palpable, they were treated with KPT-9274 and/or anti-programmed cell death 1 (PDCD1; PD1) antibody for 21 days. Tumors were measured and then removed at animal euthanasia for subsequent studies. RESULTS: We demonstrate a significant decrease in allograft growth with the combination treatment of KPT-9274 and anti-PD1 antibody without significant weight loss by the animals. This is associated with decreased (MOUSE) Naprt expression, indicating dependence of these tumors on NAMPT in parallel to what we have observed in human RCC. Histology of the tumors showed substantial necrosis regardless of treatment condition, and flow cytometry of antibody-stained tumor cells revealed that the enhanced therapeutic effect of KPT-9274 and anti-PD1 antibody was not driven by infiltration of T cells into tumors. CONCLUSIONS: This study highlights the potential of the RENCA model for evaluating immunologic responses to KPT-9274 and checkpoint inhibitor (CPI) and suggests that therapy with this combination could improve efficacy in RCC beyond what is achievable with CPI alone.

Genomic and phenotypic analyses of six offspring of a genome-edited hornless bull.

Genome editing followed by reproductive cloning was previously used to produce two hornless dairy bulls. We crossed one genome-edited dairy bull, homozygous for the dominant PC Celtic POLLED allele, with horned cows (pp) and obtained six heterozygous (PCp) polled calves. The calves had no horns and were otherwise healthy and phenotypically unremarkable. We conducted whole-genome sequencing of all animals using an Illumina HiSeq4000 to achieve ~20× coverage. Bioinformatics analyses revealed the bull was a compound heterozygote, carrying one naturally occurring PC Celtic POLLED allele and an allele containing an additional introgression of the homology-directed repair donor plasmid along with the PC Celtic allele. These alleles segregated in the offspring of this bull, and inheritance of either allele produced polled calves. No other unintended genomic alterations were observed. These data can be used to inform conversations in the scientific community, with regulatory authorities and with the public around 'intentional genomic alterations' and future regulatory actions regarding genome-edited animals.

Plasma metabolites and lipids associate with kidney function and kidney volume in hypertensive ADPKD patients early in the disease course.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and is characterized by gradual cyst growth and expansion, increase in kidney volume with an ultimate decline in kidney function leading to end stage renal disease (ESRD). Given the decades long period of stable kidney function while cyst growth occurs, it is important to identify those patients who will progress to ESRD. Recent data from our and other laboratories have demonstrated that metabolic reprogramming may play a key role in cystic epithelial proliferation resulting in cyst growth in ADPKD. Height corrected total kidney volume (ht-TKV) accurately reflects cyst burden and predicts future loss of kidney function. We hypothesize that specific plasma metabolites will correlate with eGFR and ht-TKV early in ADPKD, both predictors of disease progression, potentially indicative of early physiologic derangements of renal disease severity. METHODS: To investigate the predictive role of plasma metabolites on eGFR and/or ht-TKV, we used a non-targeted GC-TOF/MS-based metabolomics approach on hypertensive ADPKD patients in the early course of their disease. Patient data was obtained from the HALT-A randomized clinical trial at baseline including estimated glomerular filtration rate (eGFR) and measured ht-TKV. To identify individual metabolites whose intensities are significantly correlated with eGFR and ht-TKV, association analyses were performed using linear regression with each metabolite signal level as the primary predictor variable and baseline eGFR and ht-TKV as the continuous outcomes of interest, while adjusting for covariates. Significance was determined by Storey's false discovery rate (FDR) q-values to correct for multiple testing. RESULTS: Twelve metabolites significantly correlated with eGFR and two triglycerides significantly correlated with baseline ht-TKV at FDR q-value < 0.05. Specific significant metabolites, including pseudo-uridine, indole-3-lactate, uric acid, isothreonic acid, and creatinine, have been previously shown to accumulate in plasma and/or urine in both diabetic and cystic renal diseases with advanced renal insufficiency. CONCLUSIONS: This study identifies metabolic derangements in early ADPKD which may be prognostic for ADPKD disease progression. CLINICAL TRIAL: HALT Progression of Polycystic Kidney Disease (HALT PKD) Study A; Clinical www.clinicaltrials.gov identifier: NCT00283686; first posted January 30, 2006, last update posted March 19, 2015.

Arginine reprogramming in ADPKD results in arginine-dependent cystogenesis.

Research into metabolic reprogramming in cancer has become commonplace, yet this area of research has only recently come of age in nephrology. In light of the parallels between cancer and autosomal dominant polycystic kidney disease (ADPKD), the latter is currently being studied as a metabolic disease. In clear cell renal cell carcinoma (RCC), which is now considered a metabolic disease, we and others have shown derangements in the enzyme arginosuccinate synthase 1 (ASS1), resulting in RCC cells becoming auxotrophic for arginine and leading to a new therapeutic paradigm involving reducing extracellular arginine. Based on our earlier finding that glutamine pathways are reprogrammed in ARPKD, and given the connection between arginine and glutamine synthetic pathways via citrulline, we investigated the possibility of arginine reprogramming in ADPKD. We now show that, in a remarkable parallel to RCC, ASS1 expression is reduced in murine and human ADPKD, and arginine depletion results in a dose-dependent compensatory increase in ASS1 levels as well as decreased cystogenesis in vitro and ex vivo with minimal toxicity to normal cells. Nontargeted metabolomics analysis of mouse kidney cell lines grown in arginine-deficient versus arginine-replete media suggests arginine-dependent alterations in the glutamine and proline pathways. Thus, depletion of this conditionally essential amino acid by dietary or pharmacological means, such as with arginine-degrading enzymes, may be a novel treatment for this disease.

Glutamine Addiction in Kidney Cancer Suppresses Oxidative Stress and Can Be Exploited for Real-Time Imaging.

Many cancers appear to activate intrinsic antioxidant systems as a means to counteract oxidative stress. Some cancers, such as clear cell renal cell carcinoma (ccRCC), require exogenous glutamine for growth and exhibit reprogrammed glutamine metabolism, at least in part due to the glutathione pathway, an efficient cellular buffering system that counteracts reactive oxygen species and other oxidants. We show here that ccRCC xenograft tumors under the renal capsule exhibit enhanced oxidative stress compared with adjacent normal tissue and the contralateral kidney. Upon glutaminase inhibition with CB-839 or BPTES, the RCC cell lines SN12PM-6-1 (SN12) and 786-O exhibited decreased survival and pronounced apoptosis associated with a decreased GSH/GSSG ratio, augmented nuclear factor erythroid-related factor 2, and increased 8-oxo-7,8-dihydro-2'-deoxyguanosine, a marker of DNA damage. SN12 tumor xenografts showed decreased growth when treated with CB-839. Furthermore, PET imaging confirmed that ccRCC tumors exhibited increased tumoral uptake of 18F-(2S,4R)4-fluoroglutamine compared with the kidney in the orthotopic mouse model. This technique can be utilized to follow changes in ccRCC metabolism in vivo Further development of these paradigms will lead to new treatment options with glutaminase inhibitors and the utility of PET to identify and manage patients with ccRCC who are likely to respond to glutaminase inhibitors in the clinic. Cancer Res; 77(23); 6746-58. ©2017 AACR.

The Transcriptome of Estrogen-Independent Mammary Growth in Female Mice Reveals That Not All Mammary Glands Are Created Equally.

Allometric growth of ducts in the mammary glands (MGs) is widely held to be estrogen dependent. We previously discovered that the dietary fatty acid trans-10, cis-12 conjugated linoleic acid (CLA) stimulates estrogen-independent allometric growth and terminal end bud formation in ovariectomized mice. Given the similar phenotype induced by estrogen and CLA, we investigated the shared and/or divergent mechanisms underlying these changes. We confirmed MG growth induced by CLA is temporally distinct from that elicited by estrogen. We then used RNA sequencing to compare the transcriptome of the MG during similar proliferative and morphological states. Both estrogen and CLA affected the genes involved in proliferation. The transcriptome for estrogen-treated mice included canonical estrogen-induced genes, including Pgr, Areg, and Foxa1. In contrast, their expression was unchanged by CLA. However, CLA, but not estrogen, altered expression of a unique set of inflammation-associated genes, consistent with stromal changes. This CLA-altered signature included increased expression of epidermal growth factor receptor (EGFR) pathway components, consistent with the demonstration that CLA-induced MG growth is EGFR dependent. Our findings highlight a unique role for diet-induced inflammation that underlies estrogen-independent MG development.

Pathobiology of the 129:Stat1 -/- mouse model of human age-related ER-positive breast cancer with an immune infiltrate-excluded phenotype.

BACKGROUND: Stat1 gene-targeted knockout mice (129S6/SvEvTac-Stat1 tm1Rds) develop estrogen receptor-positive (ER+), luminal-type mammary carcinomas at an advanced age. There is evidence for both host environment as well as tumor cell-intrinsic mechanisms to initiate tumorigenesis in this model. In this report, we summarize details of the systemic and mammary pathology at preneoplastic and tumor-bearing time points. In addition, we investigate tumor progression in the 129:Stat1 -/- host compared with wild-type 129/SvEv, and we describe the immune cell reaction to the tumors. METHODS: Mice housed and treated according to National Institutes of Health guidelines and Institutional Animal Care and Use Committee-approved methods were evaluated by histopathology, and their tissues were subjected to immunohistochemistry with computer-assisted quantitative image analysis. Tumor cell culture and conditioned media from cell culture were used to perform macrophage (RAW264.7) cell migration assays, including the 129:Stat1 -/--derived SSM2 cells as well as control Met1 and NDL tumor cells and EpH4 normal cells. RESULTS: Tumorigenesis in 129:Stat1 -/- originates from a population of FoxA1+ large oval pale cells that initially appear and accumulate along the mammary ducts in segments or regions of the gland prior to giving rise to mammary intraepithelial neoplasias. Progression to invasive carcinoma is accompanied by a marked local stromal and immune cell response composed predominantly of T cells and macrophages. In conditioned media experiments, cells derived from 129:Stat1 -/- tumors secrete both chemoattractant and chemoinhibitory factors, with greater attraction in the extracellular vesicular fraction and inhibition in the soluble fraction. The result appears to be recruitment of the immune reaction to the periphery of the tumor, with exclusion of immune cell infiltration into the tumor. CONCLUSIONS: 129:Stat1 -/- is a unique model for studying the critical origins and risk reduction strategies in age-related ER+ breast cancer. In addition, it can be used in preclinical trials of hormonal and targeted therapies as well as immunotherapies.

Anticystogenic activity of a small molecule PAK4 inhibitor may be a novel treatment for autosomal dominant polycystic kidney disease.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common hereditary renal disease with no currently available targeted therapies. Based on the established connection between ß-catenin signaling and renal ciliopathies, and on data from our and other laboratories showing striking similarities of this disease and cancer, we evaluated the use of an orally bioavailable small molecule, KPT-9274 (a dual inhibitor of the protein kinase PAK4 and nicotinamide phosphoribosyl transferase), for treatment of ADPKD. Treatment of PKD-derived cells with this compound not only reduces PAK4 steady-state protein levels and regulates ß-catenin signaling, but also inhibits nicotinamide phosphoribosyl transferase, the rate-limiting enzyme in a key NAD salvage pathway. KPT-9274 can attenuate cellular proliferation and induce apoptosis associated with a decrease in active (phosphorylated) PAK4 and ß-catenin in several Pkd1-null murine cell lines, with a less pronounced effect on the corresponding phenotypically normal cells. Additionally, KPT-9274 shows inhibition of cystogenesis in an ex vivo model of cyclic AMP-induced cystogenesis as well as in the early stage Pkd1flox/flox:Pkhd1-Cre mouse model, the latter showing confirmation of specific anti-proliferative, apoptotic, and on-target effects. NAD biosynthetic attenuation by KPT-9274, while critical for highly proliferative cancer cells, does not appear to be important in the slower growing cystic epithelial cells during cystogenesis. KPT-9274 was not toxic in our ADPKD animal model or in other cancer models. Thus, this small molecule inhibitor could be evaluated in a clinical trial as a viable therapy of ADPKD.

Trans-Fatty Acid-Stimulated Mammary Gland Growth in Ovariectomized Mice is Fatty Acid Type and Isomer Specific.

We previously reported that the trans-18:2 fatty acid trans-10, cis-12 conjugated linoleic acid (t10,c12-CLA) stimulates mammary gland development independent of estrogen and its receptor. Given the negative consequences of dietary trans-fatty acids on various aspects of human health, we sought to establish whether other trans-fatty acids could similarly induce ovary-independent mammary gland growth in mice. Prepubertal BALB/cJ mice were ovariectomized at 21 days of age then were fed diets enriched with cis-9, trans-11 CLA (c9,t11-CLA), or mixtures of trans-18:1 fatty acids supplied by partially hydrogenated sunflower, safflower, or linseed oil. The resultant mammary phenotype was evaluated 3 weeks later and compared to the growth response elicited by t10,c12-CLA, or the defined control diet. Whereas partially hydrogenated safflower oil increased mammary gland weight, none of the partially hydrogenated vegetable oils promoted mammary ductal growth. Similarly, the c9,t11-CLA supplemented diet was without effect on mammary development. Taken together, our data emphasize a unique effect of t10,c12-CLA in stimulating estrogen-independent mammary gland growth manifest as increased mammary ductal area and elongation that was not recapitulated by c9,t11-CLA or the partially hydrogenated vegetable oil diets.