Peter C. K. Leung

Mutation of FOXL2 in granulosa-cell tumors of the ovary

BACKGROUND Granulosa-cell tumors (GCTs) are the most common type of malignant ovarian sex cord-stromal tumor (SCST). The pathogenesis of these tumors is unknown. Moreover, their histopathological diagnosis can be challenging, and there is no curative treatment beyond surgery. METHODS We analyzed four adult-type GCTs using whole-transcriptome paired-end RNA sequencing. We identified putative GCT-specific mutations that were present in at least three of these samples but were absent from the transcriptomes of 11 epithelial ovarian tumors, published human genomes, and databases of single-nucleotide polymorphisms. We confirmed these variants by direct sequencing of complementary DNA and genomic DNA. We then analyzed additional tumors and matched normal genomic DNA, using a combination of direct sequencing, analyses of restriction-fragment-length polymorphisms, and TaqMan assays. RESULTS All four index GCTs had a missense point mutation, 402C-->G (C134W), in FOXL2, a gene encoding a transcription factor known to be critical for granulosa-cell development. The FOXL2 mutation was present in 86 of 89 additional adult-type GCTs (97%), in 3 of 14 thecomas (21%), and in 1 of 10 juvenile-type GCTs (10%). The mutation was absent in 49 SCSTs of other types and in 329 unrelated ovarian or breast tumors. CONCLUSIONS Whole-transcriptome sequencing of four GCTs identified a single, recurrent somatic mutation (402C-->G) in FOXL2 that was present in almost all morphologically identified adult-type GCTs. Mutant FOXL2 is a potential driver in the pathogenesis of adult-type GCTs.

Estrogen regulates snail and slug in the down-regulation of E-cadherin and induces metastatic potential of ovarian cancer cells through estrogen receptor α

Tumorigenesis is a multistep process involving dysregulated cell growth and metastasis. Considerable evidence implicates a mitogenic action of estrogen in early ovarian carcinogenesis. In contrast, its influence in the metastatic cascade of ovarian tumor cells remains obscure. In the present study, we showed that 17beta-estradiol (E2) increased the metastatic potential of human epithelial ovarian cancer cell lines. E2 treatment led to clear morphological changes characteristic of epithelial-mesenchymal transition (EMT) and an enhanced cell migratory propensity. These morphological and functional alterations were associated with changes in the abundance of EMT-related genes. Upon E2 stimulation, expression and promoter activity of the epithelial marker E-cadherin were strikingly suppressed, whereas EMT-associated transcription factors, Snail and Slug, were significantly up-regulated. This up-regulation was attributed to the increase in gene transcription activated by E2. Depletion of endogenous Snail or Slug using small interfering RNA (siRNA) attenuated E2-mediated decrease in E-cadherin. In addition, E2-induced cell migration was also neutralized by the siRNAs, suggesting that both transcription factors are indispensable for the prometastatic actions of E2. More importantly, by using selective estrogen receptor (ER) agonists, forced expression, and siRNA approaches, we identified that E2 triggered the metastatic behaviors exclusively through an ERalpha-dependent pathway. We also showed that ERbeta had an opposing action on ERalpha because the presence of ERbeta completely inhibited the EMT and down-regulation of E-cadherin induced by ERalpha. Collectively, this study provides a compelling argument that estrogen can potentiate tumor progression by EMT induction and highlights the crucial role of ERalpha in ovarian tumorigenesis.

Transgenerational Glucose Intolerance With Igf2/H19 Epigenetic Alterations in Mouse Islet Induced by Intrauterine Hyperglycemia

Gestational diabetes mellitus (GDM) has been shown to be associated with high risk of diabetes in offspring. However, the mechanisms involved and the possibilities of transgenerational transmission are still unclear. We intercrossed male and female adult control and first-generation offspring of GDM (F1-GDM) mice to obtain the second-generation (F2) offspring in four groups: C♂-C♀, C♂-GDM♀, GDM♂-C♀, and GDM♂-GDM♀. We found that birth weight significantly increased in F2 offspring through the paternal line with impaired glucose tolerance (IGT). Regardless of birth from F1-GDM with or without IGT, high risk of IGT appeared as early as 3 weeks in F2 offspring and progressed through both parental lineages, especial the paternal line. IGT in male offspring was more obvious than that in females, with parental characteristics and sex-specific transmission. In both F1 and F2 offspring of GDM, the expression of imprinted genes Igf2 and H19 was downregulated in pancreatic islets, caused by abnormal methylation status of the differentially methylated region, which may be one of the mechanisms for impaired islet ultrastructure and function. Furthermore, altered Igf2 and H19 gene expression was found in sperm of adult F1-GDM, regardless of the presence of IGT, indicating that changes of epigenetics in germ cells contributed to transgenerational transmission.

Role of gonadotropin-releasing hormone as an autocrine growth factor in human ovarian surface epithelium

Epithelial ovarian cancer, which accounts for 80–90% of all ovarian cancers, is the most common cause of death from gynecological malignancies and is believed to originate from the ovarian surface epithelium. In the present study we investigated the expression of GnRH and its receptor in human ovarian surface epithelial (hOSE) cells and provided novel evidence that GnRH may have antiproliferative effects in this tissue. Using RT-PCR and Southern blot analysis, we cloned the GnRH and GnRH receptor (GnRHR) in hOSE cells. Sequence analysis revealed that GnRH and its receptor have sequences identical to those found in the hypothalamus and pituitary, respectively. To address whether GnRH regulates its own and receptor messenger RNA (mRNA), the cells were treated with different concentrations of the GnRH agonist (d-Ala6)-GnRH. Expression levels of GnRH and its receptor were investigated using quantitative and competitive RT-PCR, respectively. Interestingly, a biphasic effect was observed for the GnRH and GnRHR ...

Constitutive and conditional cadherin expression in cultured human ovarian surface epithelium: Influence of family history of ovarian cancer

Epithelial ovarian carcinomas arise in a simple mesothelium (ovarian surface epithelium, OSE) but exhibit properties of oviductal and endometrial epithelia. Thus, during malignant progression, their differentiation proceeds from simple to complex, in contrast to carcinomas in other tissues. Related changes in OSE of women with a history of familial ovarian cancer indicate that this aberrant differentiation is initiated very early in neoplastic progression. The mechanisms underlying this process are not understood. Because cadherins are known regulators of differentiation, we investigated the relationship of the cadherins E, N and P to OSE morphology, growth patterns and differentiation in cultures of normal and metaplastic OSE from women with (FH‐OSE) and without (NFH‐OSE) a family history of ovarian cancer and in the ovarian carcinoma lines OVCAR‐3 and CaOV3. We used immunofluorescence, RT‐PCR, in situ hybridization and Western blotting. Our results define N‐cadherin as the constitutively expressed cadherin of normal and metaplastic OSE and indicate that P‐cadherin is undetectable while E‐cadherin expression is conditional and related to genotype, stage of neoplastic progression and growth pattern. The altered expression of E‐cadherin in apparently normal OSE of women with hereditary ovarian cancer syndromes in conjunction with the known capacity of E‐cadherin to induce epithelial characteristics implicates this adhesion molecule as a possible inducer of the aberrant Mullerian differentiation which characterizes epithelial ovarian carcinomas. Abnormal differentiation in such (pre)‐neoplastic tissues may represent an early, irreversible, non‐mutational step in ovarian epithelial neoplastic progression. Int. J. Cancer 81:180–188, 1999.

Long-term growth and steroidogenic potential of human granulosa-lutein cells immortalized with SV40 large T antigen

Studies of human ovarian granulosa cells have been limited by the small numbers and short life span in culture of cells currently obtainable from clinical material. Using SV40 large T antigen, we have reproducibly immortalized freshly explanted human granulosa cells obtained through an In Vitro Fertilization Program. Of 69 independently isolated clones, 17 grew progressively into lines. In the presence of 1 mM 8-Br-cyclic AMP and 50 ng/ml pregnenolone, 15 of these 17 lines secreted progesterone. Seven lines derived from two patients were analyzed in detail. The lines differed in their responsiveness to cyclic AMP and pregnenolone, and in the timing of their steroidogenic responses. In response to cyclic AMP plus pregnenolone, progesterone secretion increased up to 20-fold. Three clones tested all responded to forskolin and cholera toxin with up to 7-fold increases in progesterone secretion. One line responded inconsistently to 1 IU/ml hCG but not to FSH. The steroidogenic responses to cyclic AMP were accompanied by morphologic cell rounding. The immortalized cell lines underwent 40-60 population doublings, thus, providing a theoretical yield of up to 10(18) cells per line. These results show that a high proportion of SV40 immortalized lines is steroidogenic. These lines provide a new experimental model for studies of cell lines that appear representative of different states of differentiation of human granulosa cells.

Multi-factorial role of GnRH-I and GnRH-II in the human ovary

Normal ovarian functions are regulated by a wide variety of endocrine hormones, local paracrine and autocrine factors, which functionally interact with each other in a highly coordinated fashion. Recent findings have demonstrated that both forms of gonadotropin-releasing hormone (GnRH-I and GnRH-II) are expressed in various compartments of the human ovary including the granulosa-luteal cells, ovarian surface epithelial cells and ovarian tumors, and their expressions have been shown to be tightly regulated by gonadal steroids and gonadotropins. Functionally, these neuropeptides exert diverse biological effects in the ovary via binding to their cognate receptors, supporting the notion that these peptides act as paracrine and autocrine factors in modulating local ovarian functions. In this review, we will summarize recent literatures regarding the regulation of GnRH-I and GnRH-II gene expressions in the human ovary, and discuss the possible signal transduction mechanisms by which these hormones exert their actions in the gonad. Recent cloning of the second form of the GnRH receptor (GnRH-II receptor) in primates and other vertebrates demonstrated that it was structurally, and thus, functionally distinct from the GnRH-I receptor. Cell proliferation studies showed that GnRH-II inhibited the growth of human ovarian cancer cells that express GnRH-II but not GnRH-I receptor, indicating that the GnRH-II binding sites are functional in these cells. However, it remains unknown if GnRH-II receptor is expressed as a full-length, properly processed and functional gene transcript in humans, and its potential physiological roles such as differential regulation of gonadotropin secretion, neuroendocrine modulation and female sexual behavior await further investigation.

Progesterone Receptor (PR) Isoforms PRA and PRB Differentially Regulate Expression of the Breast Cancer Resistance Protein in Human Placental Choriocarcinoma BeWo Cells

Breast cancer resistance protein (BCRP) plays a significant role in drug disposition and in conferring multidrug resistance in cancer cells. Previous studies have shown that steroid hormones such as 17β-estradiol and progesterone can affect BCRP expression in cancer cells. In this study, we investigated the molecular mechanism by which BCRP expression in human placental choriocarcinoma BeWo cells is regulated by progesterone. Transfection of the progesterone receptor (PR) isoforms PRA and PRB resulted in a similarly increased expression of PRA and PRB, respectively. However, progesterone significantly increased BCRP expression and activity only in PRB-transfected cells. This stimulatory effect of progesterone was abrogated by the PR antagonist mifepristone (RU-486). Consistently, transcriptional activity of the BCRP promoter was induced 2- to 6-fold by 10-8 to 10-5 M progesterone in PRB-transfected cells. Progesterone had little effect on BCRP expression and activity and transcriptional activity of the BCRP promoter in PRA-transfected cells; however, cotransfection of PRA and PRB significantly decreased the progesterone-response compared with that in cells transfected with only PRB. Mutations in a novel progesterone response element (PRE) identified between -243 to -115 bp of the BCRP promoter region significantly attenuated the progesterone-response in PRB-transfected cells, and deletion of the PRE nearly completely abrogated the progesterone effect. Specific binding of both PRA and PRB to the BCRP promoter through the identified PRE was confirmed using the electrophoretic mobility shift assay. Collectively, progesterone induces BCRP expression in BeWo cells via PRB but not PRA. PRA represses the PRB activity. Thus, PRA and PRB differentially regulate BCRP expression in BeWo cells.

Gonadotropin-Releasing Hormone Promotes Ovarian Cancer Cell Invasiveness through c-Jun NH2-Terminal Kinase–Mediated Activation of Matrix Metalloproteinase (MMP)-2 and MMP-9

Gonadotropin-releasing hormone (GnRH) receptor is present in 80% of ovarian cancer, and numerous studies have provided evidence for a role of GnRH in cell proliferation. In this study, the effect of GnRH on the invasion potential of ovarian cancer cells was investigated. In vitro migration and cell invasion assays with the ovarian cancer cell lines Caov-3 and OVCAR-3 revealed the biphasic nature of GnRH; low concentrations of GnRH agonist (GnRHa) increased the cell motility and invasiveness of these cells, but at increased concentrations, the stimulatory effect was insignificant. Reverse transcription-PCR, Western blot, and gelatin zymography showed that the expression of metastasis-related proteinases, matrix metalloproteinase (MMP)-2 and MMP-9, was up-regulated and activated by GnRHa. Moreover, we observed that GnRHa was able to transactivate the MMP-2 and MMP-9 promoters. The invasive/migratory phenotype activated by GnRHa can be blocked by specific inhibitors or neutralizing antibodies to MMP-2 and MMP-9. Knockdown of the GnRH receptor using small interfering RNA significantly inhibited the GnRH-induced MMP activation, invasion, and migration. In addition, we showed that the c-Jun NH(2)-terminal kinase, but not extracellular signal-regulated kinase 1/2 or p38 mitogen-activated protein kinase, signaling pathway was critical for GnRH-mediated up-regulation of MMP, cell invasion, and motility. These results indicate for the first time an expanded role for GnRH in other aspects of ovarian tumor progression, such as metastasis, via activation of MMP and the subsequent increase in cell migration and invasion.

E-cadherin inhibits tumor cell growth by suppressing PI3K/Akt signaling via β-catenin-Egr1-mediated PTEN expression.

E-cadherin is a cell–cell adhesion protein and tumor suppressor that is silenced in many malignancies. E-cadherin is thought to suppress tumor cell growth by antagonizing β-catenin signaling. However, the role of E-cadherin in ovarian cancer progression is still controversial. In this study, we showed that loss of E-cadherin induced ovarian cancer cell growth and constitutive activation of phosphoinositide 3-kinase (PI3K)/Akt signaling by the inhibition of phosphatase and tensin homolog (PTEN) transcription through the downregulation of early growth response gene 1 (Egr1). In addition, immunofluorescence microscopy and T-cell factor promoter/luciferase reporter assays showed that E-cadherin loss was associated with enhanced nuclear β-catenin signaling. Constitutive activation of PI3K/Akt signaling reinforced nuclear β-catenin signaling by inactivating glycogen synthase kinase-3β indicating cross-talk between the PI3K/Akt and β-catenin signaling pathways. Finally, we found that E-cadherin negatively regulates tumor cell growth, in part, by positively regulating PTEN expression via β-catenin-mediated Egr1 regulation, thus influencing PI3K/Akt signaling. In summary, endogenous E-cadherin inhibits PI3K/Akt signaling by antagonizing β-catenin-Egr1-mediated repression of PTEN expression. Thus, the loss of E-cadherin itself may contribute to dysregulated PI3K/Akt signaling through its effects on PTEN, or it may exacerbate the frequent activation of PI3K/Akt signaling that occurs as a result of overexpression, mutation and/or amplification.