Polyamine Pathway Inhibitor DENSPM Suppresses Lipid Metabolism in Pheochromocytoma Cell Line.

Pheochromocytomas (PCCs) are tumors arising from chromaffin cells in the adrenal medulla, and paragangliomas (PGLs) are tumors derived from extra-adrenal sympathetic or parasympathetic paraganglia; these tumors are collectively referred to as PPGL cancer. Treatment for PPGL primarily involves surgical removal of the tumor, and only limited options are available for treatment of the disease once it becomes metastatic. Human carriers of the heterozygous mutations in the succinate dehydrogenase subunit B (SDHB) gene are susceptible to the development of PPGL. A physiologically relevant PCC patient-derived cell line hPheo1 was developed, and SDHB_KD cells carrying a stable short hairpin knockdown of SDHB were derived from it. An untargeted metabolomic approach uncovered an overactive polyamine pathway in the SDHB_KD cells that was subsequently fully validated in a large set of human SDHB-mutant PPGL tumor samples. We previously reported that treatment with the polyamine metabolism inhibitor N1,N11-diethylnorspermine (DENSPM) drastically inhibited growth of these PCC-derived cells in culture as well as in xenograft mouse models. Here we explored the mechanisms underlying DENSPM action in hPheo1 and SDHB_KD cells. Specifically, by performing an RNAseq analysis, we have identified gene expression changes associated with DENSPM treatment that broadly interfere with all aspects of lipid metabolism, including fatty acid (FA) synthesis, desaturation, and import/uptake. Furthermore, by performing an untargeted lipidomic liquid chromatography-mass spectrometry (LC/MS)-based analysis we uncovered specific groups of lipids that are dramatically reduced as a result of DENSPM treatment. Specifically, the bulk of plasmanyl ether lipid species that have been recently reported as the major determinants of cancer cell fate are notably decreased. In summary, this work suggests an intersection between active polyamine and lipid pathways in PCC cells.

Nicotine Exposure during Rodent Pregnancy Alters the Composition of Maternal Gut Microbiota and Abundance of Maternal and Amniotic Short Chain Fatty Acids.

Tobacco smoking is the leading cause of preventable death. Numerous reports link smoking in pregnancy with serious adverse outcomes, such as miscarriage, stillbirth, prematurity, low birth weight, perinatal morbidity, and infant mortality. Corollaries of consuming nicotine in pregnancy, separate from smoking, are less explored, and the mechanisms of nicotine action on maternal-fetal communication are poorly understood. This study examined alterations in the maternal gut microbiome in response to nicotine exposure during pregnancy. We report that changes in the maternal gut microbiota milieu are an important intermediary that may mediate the prenatal nicotine exposure effects, affect gene expression, and alter fetal exposure to circulating short-chain fatty acids (SCFAs) and leptin during in utero development.

Targeting pheochromocytoma/paraganglioma with polyamine inhibitors.

BACKGROUND: Pheochromocytomas (PCCs) and paragangliomas (PGLs) are neuroendocrine tumors that are mostly benign. Metastatic disease does occur in about 10% of cases of PCC and up to 25% of PGL, and for these patients no effective therapies are available. Patients with mutations in the succinate dehydrogenase subunit B (SDHB) gene tend to have metastatic disease. We hypothesized that a down-regulation in the active succinate dehydrogenase B subunit should result in notable changes in cellular metabolic profile and could present a vulnerability point for successful pharmacological targeting. METHODS: Metabolomic analysis was performed on human hPheo1 cells and shRNA SDHB knockdown hPheo1 (hPheo1 SDHB KD) cells. Additional analysis of 115 human fresh frozen samples was conducted. In vitro studies using N1,N11-diethylnorspermine (DENSPM) and N1,N12- diethylspermine (DESPM) treatments were carried out. DENSPM efficacy was assessed in human cell line derived mouse xenografts. RESULTS: Components of the polyamine pathway were elevated in hPheo1 SDHB KD cells compared to wild-type cells. A similar observation was noted in SDHx PCC/PGLs tissues compared to their non-mutated counterparts. Specifically, spermidine, and spermine were significantly elevated in SDHx-mutated PCC/PGLs, with a similar trend in hPheo1 SDHB KD cells. Polyamine pathway inhibitors DENSPM and DESPM effectively inhibited growth of hPheo1 cells in vitro as well in mouse xenografts. CONCLUSIONS: This study demonstrates overactive polyamine pathway in PCC/PGL with SDHB mutations. Treatment with polyamine pathway inhibitors significantly inhibited hPheo1 cell growth and led to growth suppression in xenograft mice treated with DENSPM. These studies strongly implicate the polyamine pathway in PCC/PGL pathophysiology and provide new foundation for exploring the role for polyamine analogue inhibitors in treating metastatic PCC/PGL. PRéCIS: Cell line metabolomics on hPheo1 cells and PCC/PGL tumor tissue indicate that the polyamine pathway is activated. Polyamine inhibitors in vitro and in vivo demonstrate that polyamine inhibitors are promising for malignant PCC/PGL treatment. However, further research is warranted.

Adrenal Development in Mice Requires GATA4 and GATA6 Transcription Factors.

The adrenal glands consist of an outer cortex and an inner medulla, and their primary purposes include hormone synthesis and secretion. The adrenal cortex produces a complex array of steroid hormones, whereas the medulla is part of the sympathetic nervous system and produces the catecholamines epinephrine and norepinephrine. In the mouse, GATA binding protein (GATA) 4 and GATA6 transcription factors are coexpressed in several embryonic tissues, including the adrenal cortex. To explore the roles of GATA4 and GATA6 in mouse adrenal development, we conditionally deleted these genes in adrenocortical cells using the Sf1Cre strain of animals. We report here that mice with Sf1Cre-mediated double deletion of Gata4 and Gata6 genes lack identifiable adrenal glands, steroidogenic factor 1-positive cortical cells and steroidogenic gene expression in the adrenal location. The inactivation of the Gata6 gene alone (Sf1Cre;Gata6(flox/flox)) drastically reduced the adrenal size and corticosterone production in the adult animals. Adrenocortical aplasia is expected to result in the demise of the animal within 2 weeks after birth unless glucocorticoids are provided. In accordance, Sf1Cre;Gata4(flox/flox)Gata6(flox/flox) females depend on steroid supplementation to survive after weaning. Surprisingly, Sf1Cre;Gata4(flox/flox)Gata6(flox/flox) males appear to live normal lifespans as vital steroidogenic synthesis shifts to their testes. Our results reveal a requirement for GATA factors in adrenal development and provide a novel tool to characterize the transcriptional network controlling adrenocortical cell fates.

Combined loss of the GATA4 and GATA6 transcription factors in male mice disrupts testicular development and confers adrenal-like function in the testes.

The roles of the GATA4 and GATA6 transcription factors in testis development were examined by simultaneously ablating Gata4 and Gata6 with Sf1Cre (Nr5a1Cre). The deletion of both genes resulted in a striking testicular phenotype. Embryonic Sf1Cre; Gata4(flox/flox) Gata6(flox/flox) (conditional double mutant) testes were smaller than control organs and contained irregular testis cords and fewer gonocytes. Gene expression analysis revealed significant down-regulation of Dmrt1 and Mvh. Surprisingly, Amh expression was strongly up-regulated and remained high beyond postnatal day 7, when it is normally extinguished. Neither DMRT1 nor GATA1 was detected in the Sertoli cells of the mutant postnatal testes. Furthermore, the expression of the steroidogenic genes Star, Cyp11a1, Hsd3b1, and Hsd17b3 was low throughout embryogenesis. Immunohistochemical analysis revealed a prominent reduction in cytochrome P450 side-chain cleavage enzyme (CYP11A1)- and 3ß-hydroxysteroid dehydrogenase-positive (3ßHSD) cells, with few 17α-hydroxylase/17,20 lyase-positive (CYP17A1) cells present. In contrast, in postnatal Sf1Cre; Gata4(flox/flox) Gata6(flox/flox) testes, the expression of the steroidogenic markers Star, Cyp11a1, and Hsd3b6 was increased, but a dramatic down-regulation of Hsd17b3, which is required for testosterone synthesis, was observed. The genes encoding adrenal enzymes Cyp21a1, Cyp11b1, Cyp11b2, and Mcr2 were strongly up-regulated, and clusters containing numerous CYP21A2-positive cells were localized in the interstitium. These data suggest a lack of testis functionality, with a loss of normal steroidogenic testis function, concomitant with an expansion of the adrenal-like cell population in postnatal conditional double mutant testes. Sf1Cre; Gata4(flox/flox) Gata6(flox/flox) animals of both sexes lack adrenal glands; however, despite this deficiency, males are viable in contrast to the females of the same genotype, which die shortly after birth.

Teratoma formation leads to failure of treatment for type I diabetes using embryonic stem cell-derived insulin-producing cells.

Embryonic stem (ES) cells have been proposed to be a powerful tool in the study of pancreatic disease, as well as a potential source for cell replacement therapy in the treatment of diabetes. However, data demonstrating the feasibility of using pancreatic islet-like cells differentiated from ES cells remain controversial. In this study we characterized ES cell-derived insulin-expressing cells and assessed their suitability for the treatment of type I diabetes. ES cell-derived insulin-stained cell clusters expressed insulin mRNA and transcription factors associated with pancreatic development. The majority of insulin-positive cells in the clusters also showed immunoreactivity for C-peptide. Insulin was stored in the cytoplasm and released into the culture medium in a glucose-dependent manner. When the cultured cells were transplanted into diabetic mice, they reversed the hyperglycemic state for approximately 3 weeks, but the rescue failed due to immature teratoma formation. Our studies demonstrate that reversal of hyperglycemia by transplantation of ES cell-derived insulin-producing cells is possible. However, the risk of teratoma formation would need to be eliminated before ES cell-based therapies for the treatment of diabetes are considered.

Adult bone marrow-derived cells trans-differentiating into insulin-producing cells for the treatment of type I diabetes.

Recent findings suggest that bone marrow (BM) cells have the capacity to differentiate into a variety of cell types including endocrine cells of the pancreas. We report that BM derived cells, when cultured under defined conditions, were induced to trans-differentiate into insulin-producing cells. Furthermore, these insulin-producing cells formed aggregates that, upon transplantation into mice, acquired architecture similar to islets of Langerhans. These aggregates showed endocrine gene expression for insulin (I and II), glucagon, somatostatin and pancreatic polypeptide. Immunohistochemistry also confirmed that these aggregates were positive for insulin, somatostatin, pancreatic polypeptide and C-peptide. Also, Western and ELISA analysis demonstrated expression of proinsulin and/or secretion of active insulin upon glucose challenge. Subcapsular renal transplantation of these aggregates into hyperglycemic mice lowered circulating blood glucose levels and maintained comparatively normal glucose levels for up to 90 days post-transplantation. Graft removal resulted in rapid relapse and death in experimental animals. In addition, electron microscopy revealed these aggregates had acquired ultrastructure typically associated with mature beta (beta) cells. These results demonstrate that adult BM cells are capable of trans-differentiating into a pancreatic lineage in vitro and may represent a pool of cells for the treatment of diabetes mellitus.

Hepatic oval 'stem' cell in liver regeneration.

Hepatic oval cell activation, proliferation, and differentiation has been observed under certain physiological conditions, mainly when the proliferation of existing hepatocytes has been inhibited followed by severe hepatic injury. Hepatic oval cells display a distinct phenotype and have been shown to be a bipotential progenitor of two types of epithelial cells found in the liver, hepatocytes and bile ductular cells. Bone marrow stem cells have recently been shown to be a potential source of the hepatic oval cells and that reconstitution of an injured liver from a purified stem cell population is possible. The focus of this review is on the studies involving the activation, proliferation, and differentiation of these hepatic oval cells and the role that they play in regeneration of the damaged liver. In order to present the potentiality of the hepatic oval cell, an experimental model that involves the inhibition of normal hepatic growth and division as well as severe hepatic injury via chemical or surgical means has been employed. In this model, an as yet undetermined signal or perhaps the lack of regenerative capability in the hepatocytes activates the hepatic oval cell compartment. However, other than understanding a potential origin of these cells and some of the markers that characterize them, it still remains unclear as to how these cells migrate ('home') into the damaged areas and how they begin their differentiation into mature and functioning hepatic cells.

SDF-1alpha/CXCR4: a mechanism for hepatic oval cell activation and bone marrow stem cell recruitment to the injured liver of rats.

Stromal derived factor-1 alpha (SDF-1alpha) and its receptor CXCR4 have been shown to play a role in the systematic movement of hematopoietic stem cells (HSC) in the fetal and adult stages of hematopoiesis. Under certain physiological conditions liver oval cells can participate in the regeneration of the liver. We have shown that a percentage of oval cells are of hematopoietic origin. Others have shown that bone marrow derived stem cells can participate in liver regeneration as well. In this study we examined the role of SDF-1alpha and its receptor CXCR4 as a possible mechanism for oval cell activation in oval cell aided liver regeneration. In massive liver injury models where oval cell repair is involved hepatocytes up-regulate the expression of SDF-1alpha, a potent chemoattractant for hematopoietic cells. However, when moderate liver injury occurs, proliferation of resident hepatocytes repairs the injury. Under these conditions SDF-1alpha expression is not up-regulated and oval cells are not activated in the liver. In addition, we show that oval cells express CXCR4, the only known receptor for SDF-1alpha. Lastly, in vitro chemotaxis assays demonstrated that oval cells migrate along a SDF-1alpha gradient which suggests that the SDF-1alpha/CXCR4 interaction is a mechanism by which the oval cell compartment could be activated and possibly recruit a second wave of bone marrow stem cells to the injured liver. In conclusion, these experiments begin to shed light on a possible mechanism, which may someday lead to a better understanding of the hepatic and hematopoietic interaction in oval cell aided liver regeneration.