Ruth M. Escalona

Pituitary cell lines and their endocrine applications

The pituitary gland is an important component of the endocrine system, and together with the hypothalamus, exerts considerable influence over the functions of other endocrine glands. The hypothalamus either positively or negatively regulates hormonal productions in the pituitary through its release of various trophic hormones which act on specific cell types in the pituitary to secrete a variety of pituitary hormones that are important for growth and development, metabolism, reproductive and nervous system functions. The pituitary is divided into three sections-the anterior lobe which constitute the majority of the pituitary mass and is composed primarily of five hormone-producing cell types (thyrotropes, lactotropes, corticotropes, somatotropes and gonadotropes) each secreting thyrotropin, prolactin, ACTH, growth hormone and gonadotropins (FSH and LH) respectively. There is also a sixth cell type in the anterior lobe-the non-endocrine, agranular, folliculostellate cells. The intermediate lobe produces melanocyte-stimulating hormone and endorphins, whereas the posterior lobe secretes anti-diuretic hormone (vasopressin) and oxytocin. Representative cell lines of all the six cell types of the anterior pituitary have been established and have provided valuable information on genealogy of the various cell lineages, endocrine feedback control of hormone synthesis and secretions, intrapituitary interactions between the various cell types, as well as the role of specific transcription factors that determine each differentiated cell phenotype. In this review, we will discuss the morphology and function of the cell types that make up the anterior pituitary, and the characteristics of the various functional anterior pituitary cell systems that have been established to be representative of each anterior pituitary cell lineage.

Fetal Testis Dysgenesis and Compromised Leydig Cell Function in Tgfbr3 (Betaglycan) Knockout Mice

Abstract Betaglycan (Tgfbr3) is a coreceptor for transforming growth factor-beta (TGFB) superfamily ligands. In the current study, a defect in seminiferous cord formation was detected in 12.5–13.5 days postcoitum (dpc) betaglycan null murine testis. Immunohistochemistry with antibodies against cell-specific markers revealed defects in somatic cell populations. To confirm these data, quantitative real-time PCR was performed to determine changes in the expression levels of genes involved in fetal testis cell differentiation and function. The expression levels of the Leydig cell markers Insl3, Cyp17a1, Cyp11a1, Star, and Hsd3b1 were reduced in knockout testis compared to wild-type testis, beginning at 12.5 dpc. Whole mount in situ hybridization confirmed that Cyp11a1 expression was reduced in the null testis, but its distribution pattern was unchanged. Apoptosis was not affected by the loss of betaglycan, but proliferation within the interstitium was reduced at 14.5 dpc. However, morphometric analysis showed no changes in Leydig cell counts between the wild-type and the knockout testes at 14.5 dpc, indicating that fetal Leydig function, rather than number, was affected by the loss of betaglycan. The expression levels of Sertoli cell markers Dhh, Sox9, and Amh were also reduced in the knockout testis at 14.5 dpc. However, the expression of fetal germ cell markers Pou5f1 and DDX4 were not changed across the genotypes at any age examined. Our data show that the presence of betaglycan is required for normal cord formation, normal fetal Leydig cell development, and the establishment of fetal testis endocrine function, thus implicating TGFB superfamily members as regulators of early fetal testis structure and function.

Loss of Betaglycan Contributes to the Malignant Properties of Human Granulosa Tumor Cells

Betaglycan is a type III TGFbeta receptor that modulates cellular sensitivity to inhibins and TGFbeta. Previous studies have suggested that betaglycan acts as a tumor suppressor in certain human epithelial cancers. However, the roles of betaglycan in ovarian granulosa cell tumors (GCTs) are poorly understood. The objective of this study was to determine whether human GCTs exhibit betaglycan expression and, if so, what impact this receptor has on tumor biology. Real-time PCR was used to quantify betaglycan transcripts in human GCTs (n = 17) and normal premenopausal ovaries (n = 11). This analysis established that GCTs exhibited a significant 2-fold lower mean betaglycan mRNA level as compared with the normal ovary (P < 0.05). Similarly, two human GCT cell lines, KGN and COV434, exhibited low betaglycan expression and poor responsiveness to TGFbeta and inhibin A in luciferase reporter assays, which was restored by stable transfection of wild-type betaglycan. Betaglycan significantly increased the adhesion of COV434 (P < 0.05) and KGN (P < 0.0001) cells, decreased cellular invasion through Matrigel, and inhibited wound healing. Expression of mutant forms of betaglycan that are defective in TGFbeta and/or inhibin binding in each GCT cell line revealed that the inhibitory effects of betaglycan on wound healing were most strongly linked to the inhibin-binding region of betaglycan. Furthermore, knockdown of INHA mRNA expression abrogated the betaglycan-mediated inhibition of wound healing and invasion, whereas both INHA silencing and TGFbeta neutralization abolished the betaglycan-mediated increase in adhesion to substrate. These data suggest that loss of betaglycan contributes to the pathogenesis of GCTs.

Targeted Disruption of the JAK2/STAT3 Pathway in Combination with Systemic Administration of Paclitaxel Inhibits the Priming of Ovarian Cancer Stem Cells Leading to a Reduced Tumor Burden

Chemotherapy resistance associated with recurrent disease is the major cause of poor survival of ovarian cancer patients. We have recently demonstrated activation of the JAK2/STAT3 pathway and the enhancement of a cancer stem cell (CSC)-like phenotype in ovarian cancer cells treated in vitro with chemotherapeutic agents. To elucidate further these mechanisms in vivo, we used a two-tiered paclitaxel treatment approach in nude mice inoculated with ovarian cancer cells. In the first approach, we demonstrate that a single intraperitoneal administration of paclitaxel in mice 7 days after subcutaneous transplantation of the HEY ovarian cancer cell line resulted in a significant increase in the expression of CA125, Oct4, and CD117 in mice xenografts compared to control mice xenografts which did not receive paclitaxel. In the second approach, mice were administered once weekly with paclitaxel and/or a daily dose of the JAK2-specific inhibitor, CYT387, over 4 weeks. Mice receiving paclitaxel only demonstrated a significant decrease in tumor volume compared to control mice. At the molecular level, mouse tumors remaining after paclitaxel administration showed a significant increase in the expression of Oct4 and CD117 coinciding with a significant activation of the JAK2/STAT3 pathway compared to control tumors. The addition of CYT387 with paclitaxel resulted in the suppression of JAK2/STAT3 activation and abrogation of Oct4 and CD117 expression in mouse xenografts. This coincided with significantly smaller tumors in mice administered CYT387 in addition to paclitaxel, compared to the control group and the group of mice receiving paclitaxel only. These data suggest that the systemic administration of paclitaxel enhances Oct4- and CD117-associated CSC-like marker expression in surviving cancer cells in vivo, which can be suppressed by the addition of the JAK2-specific inhibitor CYT387, leading to a significantly smaller tumor burden. These novel findings have the potential for the development of CSC-targeted therapy to improve the treatment outcomes of ovarian cancer patients.

Prostaglandin E and F receptor expression and myometrial sensitivity at labor onset in the sheep

Abstract Prostaglandins (PGs) play a pivotal role in the initiation and progression of term and preterm labor. Uterine activity is stimulated primarily by PGE2 and PGF2α acting on prostaglandin E (EP) and prostaglandin F (FP) receptors, respectively. Activation of FP receptors strongly stimulates the myometrium, whereas stimulation of EP receptors may lead to contraction or relaxation, depending on the EP subtype (EP1–4) expression. Thus, the relative expression of FP and EP1–4 may determine the responsiveness to PGE2 and PGF2α. The aims of this study were to characterize the expression of EP1–4 and FP in intrauterine tissues and placentome, together with myometrial responsiveness to PG, following the onset of dexamethasone-induced preterm and spontaneous term labor. Receptor mRNA expression was measured using quantitative real-time polymerase chain reaction using species-specific primers. There was no increase in myometrial contractile receptor expression at labor onset, nor was there a change in sensitivity to PGE2 and PGF2α. This suggests expression of these receptors reaches maximal levels by late gestation in sheep. Placental tissue showed a marked increase in EP2 and EP3 receptor expression, the functions of which are unknown at this time. Consistent with previous reports, these results suggest that PG synthesis is the main factor in the regulation of uterine contractility at labor. This is the first study to simultaneously report PG E and F receptor expression in the key gestational tissues of the sheep using species-specific primers at induced-preterm and spontaneous labor onset.

Unique proteome signature of post-chemotherapy ovarian cancer ascites-derived tumor cells

Eighty % of ovarian cancer patients diagnosed at an advanced-stage have complete remission after initial surgery and chemotherapy. However, most patients die within <5 years due to episodes of recurrences resulting from the growth of residual chemoresistant cells. In an effort to identify mechanisms associated with chemoresistance and recurrence, we compared the expression of proteins in ascites-derived tumor cells isolated from advanced-stage ovarian cancer patients obtained at diagnosis (chemonaive, CN) and after chemotherapy treatments (chemoresistant/at recurrence, CR) by using in-depth, high-resolution label-free quantitative proteomic profiling. A total of 2,999 proteins were identified. Using a stringent selection criterion to define only significantly differentially expressed proteins, we report identification of 353 proteins. There were significant differences in proteins encoding for immune surveillance, DNA repair mechanisms, cytoskeleton rearrangement, cell-cell adhesion, cell cycle pathways, cellular transport, and proteins involved with glycine/proline/arginine synthesis in tumor cells isolated from CR relative to CN patients. Pathway analyses revealed enrichment of metabolic pathways, DNA repair mechanisms and energy metabolism pathways in CR tumor cells. In conclusion, this is the first proteomics study to comprehensively analyze ascites-derived tumor cells from CN and CR ovarian cancer patients.

Effects of TGFbeta2 on Wild-Type and Tgfbr3 Knockout Mouse Fetal Testis

ABSTRACT TGFBR3 (betaglycan), a TGFbeta superfamily coreceptor, is essential for normal seminiferous cord and Leydig cell development in the fetal mouse testis and has been associated with testicular dysgenesis syndrome in men. However, the mechanisms underlying TGFBR3-regulated testis development are unclear. We tested the hypothesis that loss of Tgfbr3 compromises the functions of TGFbeta2 in the differentiating fetal testis. Analysis of expression of transcripts encoding the TGFbeta superfamily members showed a predominance of TGFbeta mRNAs during the critical window of development when testis structure is established (11.5–14.5 days postcoitum [dpc]). When cultured under basal conditions for 2 days, explants of 13.5 dpc wild-type fetal testis/mesonephros complexes exhibited structure and gene expression profiles resembling those observed in vivo between 13.5–15.5 dpc. Similarly, development of Tgfbr3 knockout testis explants recapitulated the dysgenesis and decreased somatic cell marker expression previously observed in vivo. TGFbeta2 treatment partially rescued cord development in 11.5–13.5 dpc Tgfbr3 knockout explants but did not significantly alter somatic or germ cell gene expression. In contrast, TGFbeta2 treatment of wild-type explants disrupted cord structure and significantly downregulated the somatic and steroidogenic cell markers Amh, Sf1, Star, Cyp11a, Hsd3b1, and Cyp17a1. We conclude that 1) the compromised cord development in Tgfbr3 null fetal testis is due to, at least in part, disrupted TGFbeta2 function; 2) the reduction in steroidogenesis observed in the Tgfbr3 null testis may be regulated by additional TGFBR3 ligands, rather than TGFbeta2; and 3) both cord maintenance and somatic cell development are highly sensitive to the levels of TGFbeta2.

Knockdown of stem cell regulator Oct4A in ovarian cancer reveals cellular reprogramming associated with key regulators of cytoskeleton-extracellular matrix remodelling

Oct4A is a master regulator of self-renewal and pluripotency in embryonic stem cells. It is a well-established marker for cancer stem cell (CSC) in malignancies. Recently, using a loss of function studies, we have demonstrated key roles for Oct4A in tumor cell survival, metastasis and chemoresistance in in vitro and in vivo models of ovarian cancer. In an effort to understand the regulatory role of Oct4A in tumor biology, we employed the use of an ovarian cancer shRNA Oct4A knockdown cell line (HEY Oct4A KD) and a global mass spectrometry (MS)-based proteomic analysis to investigate novel biological targets of Oct4A in HEY samples (cell lysates, secretomes and mouse tumor xenografts). Based on significant differential expression, pathway and protein network analyses, and comprehensive literature search we identified key proteins involved with biologically relevant functions of Oct4A in tumor biology. Across all preparations of HEY Oct4A KD samples significant alterations in protein networks associated with cytoskeleton, extracellular matrix (ECM), proliferation, adhesion, metabolism, epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs) and drug resistance was observed. This comprehensive proteomics study for the first time presents the Oct4A associated proteome and expands our understanding on the biological role of this stem cell regulator in carcinomas.

Tumour Microenvironment and Metabolic Plasticity in Cancer and Cancer Stem Cells: Perspectives on Metabolic and Immune Regulatory Signatures in Chemoresistant Ovarian Cancer Stem Cells

Cancer stem cells (CSCs) are a sub-population of tumour cells, which are responsible to drive tumour growth, metastasis and therapy resistance. It has recently been proposed that enhanced glucose metabolism and immune evasion by tumour cells are linked, and are modulated by the changing tumour microenvironment (TME) that creates a competition for nutrient consumption between tumour and different sub-types of cells attracted to the TME. To facilitate efficient nutrient distribution, oncogene-induced inflammatory milieu in the tumours facilitate adaptive metabolic changes in the surrounding non-malignant cells to secrete metabolites that are used as alternative nutrient sources by the tumours to sustain its increasing energy needs for growth and anabolic functions. This scenario also affects CSCs residing at the primary or metastatic niches. This review summarises recent advances in our understanding of the metabolic phenotypes of cancer cells and CSCs and how these processes are affected by the TME. We also discuss how the evolving TME modulates tumour cells and CSCs in cancer progression. Using previously described proteomic and genomic platforms, ovarian cancer cell lines and a mouse xenograft model we highlight the existence of metabolic and immune regulatory signatures in chemoresistant ovarian CSCs, and discuss how these processes may affect recurrence in ovarian tumours. We propose that progress in cancer control and eradication may depend not only on the elimination of highly chemoresistant CSCs, but also in designing novel strategies which would intervene with the tumour-promoting TME factors.

The Many Facets of Metzincins and Their Endogenous Inhibitors: Perspectives on Ovarian Cancer Progression

Approximately sixty per cent of ovarian cancer patients die within the first five years of diagnosis due to recurrence associated with chemoresistance. The metzincin family of metalloproteinases is enzymes involved in matrix remodeling in response to normal physiological changes and diseased states. Recently, there has been a mounting awareness of these proteinases and their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), as superb modulators of cellular communication and signaling regulating key biological processes in cancer progression. This review investigates the role of metzincins and their inhibitors in ovarian cancer. We propose that understanding the metzincins and TIMP biology in ovarian cancer may provide valuable insights in combating ovarian cancer progression and chemoresistance-mediated recurrence in patients.