Performing Department
Animal Sciences
Non Technical Summary
The mission of the NJAES is "To enhance the vitality, health, sustainability and overall quality of life in New Jersey by developing and delivering practical, effective solutions to current and future challenges to agriculture; fisheries; food; natural resources; environments; public health; and economic, community, and youth development." The proposed research relates to the goal of better public health. A high level of plasma prolactin, hyperprolactinemia, is known to be one of the major reasons for reproductive dysfunction such as amenorrhea, galactorrhea and infertility in women. Many of the patients with hyperprolactinemia also show prolactin-secreting pituitary adenomas (prolactinomas). While considerable gains have been made in establishing therapies for the treatment of prolactinomas, the underlying causes of prolactinomas remain poorly delineated. The idea that a woman's lifestyle choices and exposure to environmental toxins during pregnancy may affect her offspring's risk of various cancers is a newly emerging concept. Statistics from the Centers for Disease Control indicate that a significant number of women drink or binge-drink while pregnant. Therefore, the reports that rats exposed to alcohol during fetal development have increased susceptibility to hormonally induced pituitary tumors in adulthood suggest that offspring from this group of women may be at increased risk for prolactinomas. Alcohol promotion of aggressive tumors is observed in breast, prostate, pancreas, and colon tissues in human patients.This research addresses an important question of how alcohol promotes development of aggressive tumors in the pituitary. Our recent studies identified increased numbers of highly proliferative pituitary progenitor cells (pituispheres) in prenatal alcohol exposed rat pituitary tumors following estrogen administration, suggesting that alcohol may program the pituitary to increase cellular stemness. The gene Developmental Pluripotency Associated Protein 4 (DPPA4) is known to control cell stemness and act as an oncogenic factor. However, its role in aggressive prolactinoma development has never been studied. In this proposal, by employing the genome wide analysis and gene editing techniques, we study the role of theDPPA4 in establishing tumor aggressiveness in the pituitary. We also determine how alcohol epigenetically programs the pituitary to overexpress DPPA4 gene. The proposal also determines for the first time the role of DPPA4 and D2R/E2 interactions in the development of aggressive prolactinomas. The information gained from the proposed studies may lead to development of DPPA4 and D2R based therapy to control aggressive tumors in the pituitary. We believe that identification of the causes of pituitary pathology will pave the way for the development of novel therapeutic tools in the treatment and prevention of hyperprolactinemia and prolactinomas. This information should be of interest to the pharmaceutical and/or nutraceutical industry in New Jersey.
Animal Health Component
25%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
Aligning with the NJAES mission of "To enhance the vitality, health, sustainability and overall quality of life in New Jersey by developing and delivering practical, effective solutions to current and future challenges to agriculture; fisheries; food; natural resources; environments; public health; and economic, community, and youth development," this proposed research relates to the goal of better public health and it is also related to the NIFA national priority area 2: "Obesity."A high level of plasma prolactin, hyperprolactinemia, is known to be one of the major reasons for reproductive dysfunction such as amenorrhea, galactorrhea and infertility in women. A significant number of studies have shown an increased prevalence of obesity in patients with prolactinomas, although some studies have reported weight loss or weight gain after treatment of hyperprolactinemia. Many of the patients with hyperprolactinemia also show prolactin-secreting pituitary adenomas (prolactinomas). Pituitary tumors represent 10-15% of intra-cranial tumors. While most are benign and can be controlled by current therapeutics, some show a lack of sensitivity to a combination of therapies or recur during follow-up and are considered as aggressive with unclear epidemiology. For those with atypical morphological features suggestive of aggressive behavior and accounting for 2.7-15% of pituitary tumors, the World Health Organization (WHO) proposed the formulation 'atypical tumors' with 'uncertain malignancy'. Other features such as invasion can be predictive of aggressiveness. Lastly, 0.2% of pituitary tumors are associated with distant metastasis (most frequently in central nervous system, liver and bones) and with a mean survival of between 1 and 3 years.Research from ours and other laboratories has identified alcoholism as a potential contributor to the development of aggressive pituitary prolactinomas. A number of reports indicated a frequency of hyperprolactinemia in chronic alcoholic men and women. Alcohol-induced hyperprolactinemia has also been demonstrated in nonhuman primates and laboratory animals. Our work with laboratory rodents showed that ethanol increases plasma prolactin (PRL) levels and pituitary weight in cyclic female rats, and promotes estradiol (E2)-induced development of prolactinomas. Our research also identified a novel mechanism of ethanol's action involving a cross talk with the E2-responsive signaling cascade inhibiting the suppression of PRL production and cell proliferation by dopamine D2 receptor (D2R). Dopamine, a neurotransmitter, from the hypothalamus has long been known to be a physiological inhibitor of lactotropes controlling PRL section and cell growth. We showed that ethanol alters the ratio of D2R alternative splicing and reduces D2R expression in the pituitary tissue and in pituitary cells in cultures. In addition, we showed ethanol diminishes dopamine (DA) inhibition of PRL secretion from pituitary cells. Clinical studies identified decrement in PRL response to dopaminergic drugs in alcoholic patients, and reduce growth response to dopaminergic drugs in patients with aggressive prolactinomas.Alcohol promotion of aggressive tumors is observed in breast, prostate, pancreas, and colon tissues in human patients. Alcohol promotion of aggressive prolactinomas in the pituitary has been shown in the animal model of prenatal alcohol exposures. Although the mechanism by which alcohol promotes aggressiveness of tumors is not known, increasing evidence suggests that aberrant patterns of DNA methylation, an important epigenetic mechanism of transcriptional control, could be part of the pathogenetic mechanisms that lead to the development of alcohol-induced aggressive tumors. Using an animal model of prenatal alcohol exposure (PAE), which maintains stable alcohol epigenetic marks on the genome, we identified epigenetic modifications of two genes possibly involved in the development of aggressive prolactinomas under the influence of estrogen. We found that alcohol induces changes in the pituitary including elevated levels of DNA methylation, reduced levels of mRNA expression of D2R geneand increased production of microRNA-9 expression causing elevated levels of D2R alternative splicing and D2R functional loss. Recently, it has been shown that cells with D2R loss show stemness in both rat and human prolactinomas. We also obtained evidence that alcohol leads to an increase in cell stemness in the pituitary, suggesting the possibility that alcohol promotion of aggressive tumors may involve an increase in cell stemness. In this proposal, we propose to test the role of stem cell regulatory factorDevelopmental Pluripotency-Associated Protein 4 (DPPA4) in mediation of PAE effects on the development of aggressive tumor in the pituitary. Thus, the proposed studies will provide an important clue about PAE-induced epigenetic modifications in DPPA4, creating a cellular substrate vulnerable to reproductive dysfunction and pituitary tumors in adulthood. This information should be of interest to the pharmaceutical and/or nutraceutical industry in New Jersey.
Project Methods
Procedures-Study 1: To characterize the oncogenic potential of DPPA4 in pituispheres. This will be done by conducting both in vitro soft agar anchorage-independent growth assays as well as in vivo tumor formation assay in immunodeficient mice using control pituispheres, alcohol-fed offspring-derived (AF) pituispheres without any treatment, AF-derived pituispheres with knockout DPPA4 by CRISPR/Cas9. Pituispheres will be grown from AF and control pituitaries. To induce knockout of DPPA4 in pituispheres, we will use CRISPR/Cas9 methods employing a Cas9:crRNA:tracrRNA ribonucleoprotein (RNP) complex as described previously and following the IDT kit instructions (ThermoFischer). We will use both control and knockout cells to determine the cell proliferation rate (Cell Proliferation Assay Kit, Cell Signaling) and colony formation in soft agar (Cell Transformation Assay, Cell Biolabs) and cell migration and invasion (Cell migration assay, Cell Biolabs). Similarly, tumor formation in athmic NOD/scid mice will be determined for a period of 4 to 7 weeks after sc injections. Tumors formed at all injections sites in the mice will be collected and used for determination of tumor pathologies by measuring the Ki67 labeling index (>3%) and FGFR4, MMP, PTTG, p53 and PRL expression by RT-PCR.Procedures-Study 2: Examining the transformation activity of DPPA4 in human pituitary tumor cells and cancer. In order to examine whether DPPA4 can cause oncogenic transformation in human cells, human DPPA4 (hDPPA4) ORF will be amplified and cloned into the plasmid pIREShyg2 vector (Clontech, CA) using restriction enzymes in multiple cloning site of the vector. The resulting pIREShyg2-DPPA4 or mock vector will be transfected into human pituitary tumor derived cells using Lipofectamine 2000. All tissues will be obtained with patient consent, and no patient identifiers will be in any way associated with these tumor tissues lines. For each specimen we will have de-identified pathology information including clinical and hormone status. pIREShyg2-DPPA4 or mock vector will be transfected into human pituitary tumor derived cells using Lipofectamine 2000. We will use these cells to determine the cell proliferation rate, and colony formation in soft agar after 3 weeks of incubation and tumor formation in immunodeficient mice.Procedures-Study 3: Analyzing the transcriptional function of DPPA4 during oncogenic transformation. To analyze the potential transcriptional mechanisms by which DPPA4 causes cellular transformation, we will conduct expression microarray studies of RNA isolated from (1) AF-derived pituispheres, (2) control-pituispheres, (3) AF-pituispheres with DPPA4-/-, (4) AF-pituispheres with a synthetic non-targeting control. The genes up- or down-regulated at least 1.5-fold in knockout cells will be analyzed by gene ontology (GO) analysis using DAVID Bioinformatics Resources 6.7 (http://david.abcc.ncifcrf. gov/). The expression changes in various genes identified by the array data will be verified by qRT-PCR. To address this issue, we will investigate whether DPPA4 is recruited onto the targeted promoters thereby enhancing their expression. ChIPqPCR will be conducted on candidate targets arising from the array studies (e.g., KLF4, CD133 for multipotency, TRP53 and CDKN1A for P53 pathway). Chromatin samples will be prepared from pituispheres as described. 3 μg of each following antibody will be added to 300-400 μg chromatin samples: anti-mDPPA4 aa125-296 (R&D System) or Goat IgG or Rabbit IgG. SYBR green-based qPCR will be carried out using Absolute Blue QPCR Master Mix (ThermoFischer). We will further define the transcriptional activity of DPPA4 by performing promoter luciferase assay. We will clone the promoter regions of DPPA4 targeted genes in a luciferase reporter vector plasmid pGL3.0 and transfect the cells with this plasmid along with expression plasmid carrying DPPA4 ORF and measure luciferase activity. This would suggest the DPPA4 transcriptional activity of target genes.Procedures-Study 4:Determining if cell cycle regulation contributes to oncogenic transformation by DPPA4. To evaluate the mechanism by which DPPA4 increases cell proliferation, we will perform cell proliferation rate and cell cycle analysis and colony formation in (1) AF-pituispheres, (2) control-pituispheres, (3) AF-pituispheres with DPPA4+/-by CRISPR/Cas9, (4) control-pituispheres with a synthetic non-targeting control, (5) AF-pituispheres with synthetic control vectors, and (6) AF-pituispheres with β- catenin-/-. For β-catenin knockdown experiments, shRNA plasmid (sc-29210-SH) and copGFP control plasmid (sc-108083) will be used (Santa Cruz). We will determine the cell proliferation rate, cell cycle profile using flow cytometry cell cycle analysis by propidium iodide and the expression levels of several cyclins and G1/S transition genes by qRT-PCR and proteins by Western blots.Procedures-Study 5: Identification of if activated Wingless (Wnt) signaling is a mechanism of DPPA4- induced transformation in pituitary tumors. We propose to determine the role of β-catenin/wnt signaling in DPPA4 control mechanisms. We will conduct these studies by comparing the expression of EMT-related and β-catenin/wnt signaling-associated genes and proteins, the cell proliferation rate and in vitro migration and invasion abilities of AF-, control-, and AF-pituispheres with or without β-catenin deletionProcedures-Study 6: To examine whether the difference in DPPA4 gene expression is associated with changes in either the active epigenetic marker H3K4me3 or the repressive epigenetic marker H3K27me3 in the DPPA4 promoter. First, we will conduct ChIP assays with H3K4me3 and H3K27me3 antibodies. Immunoprecipitated DNA and input DNA will be subjected to qPCR with primers specific for the DPPA4 gene promoter to measure enrichment levels. The ChIP-enriched DNA samples and the input DNA samples will be analyzed by qPCR using primers specific for the DPPA4 promoters. We anticipate that the H3K4me3 levels in the DPPA4 promoter will be much higher in the AF pituispheres compared with those in controls, similar to the DPPA4 mRNA transcription levels. We also anticipate that the H3K27me3 levels will be very low in both the AF and control pituispheres. To further confirm the association between H3K4me3 levels and DPPA4 mRNA transcription levels, we will analyze the mRNA levels of these three genes in AF pituispheres and control pituispheres. We will also measure DNA methylation levels in the DPPA4 promoter using a Methylated DNA isolation assay (MeDIA)-assisted CpG microarray analysis using a whole-genome amplification kit (GenomePlex®, Sigma). We anticipate that DNA methylation levels in the DPPA4 promoter region will be very low and will not be different between the two cell types.Procedures-Study 7: We will also test whether K4 di- and trimethylated histone H3 labeling on the DPPA4 promoter is important for the proliferation and migration activity of pituispheres. To test this, we will evaluate the effects of histone deacetylase (HDAC) inhibitors, AR42 and MS- 275, which are know to upregulate H3K4 methylation marks in tumor cells and GSK J4 (Tocris) a blocker of Histone demethylases (KDMs). Lentiviruses containing rodent SET7/9 and control shRNA (Santa Cruz) will also be used to reduce H3K4me3 methylation in pituispheres according to the manufacturer's instructions. Pituispheres will be treated with histone deacetylase inhibitors or SET7 antisense or control shRNA, and analyzed for cell proliferation and migration, H3K4 trimethylation by immunohistochemical detection, accumulation of H3K4Me3 marks on the promoter DNA of DPPA4 by ChIP, and DPPA4 expression with western blot and RT-PCR. The downstream effects of histone deacetylase inhibitors or SET7 antisense treated pituispheres will be studied by analyzing the effect on wnt/β-catenin pathway signaling as described above.