Exploring the genome-wide impact of estrogen receptor alpha and estrogen receptor beta in breast and colon cancer cells

Estrogen signaling is involved in the development and progression of breast cancer and is implicated to be protective in colon cancer. Estrogenic actions are conveyed through transcriptional regulation by ligand stimulated estrogen receptors (ERα and ERβ). ERα is upregulated in most breast cancers and is responsible for the proliferative effect of estrogen. ERβ on the other hand is usually downregulated, and studies indicate an antiproliferative function. Therapies targeting ERα are available and commonly used in the treatment of breast cancer. In the normal colonic epithelia, however, ERβ is the most abundant estrogen receptor and the suggested mediator of the protective effects of estrogen in colon cancer. The role of ERβ in breast cancer and colon cancer is not well understood. Thus, exploring the genome-wide impact and contribution of both receptors in estrogen responsive cancers would substantially help to identify novel therapeutic and preventive strategies for these cancers. In Paper 1, we examined differences in transcriptional regulation between ERα and ERβ in the breast cancer cell line T47D. We could show that ERβ often exhibited an opposing effect on ERα-regulated genes within proliferation and regulation of cell cycle. We also demonstrated a set of genes only regulated by ERβ, indicating that, despite the high homology between the two receptors, there are differences in their transcriptional targets. The fact that ERβ opposed ERα indicates that ERβ activation may be of value in the treatment of breast cancer. To further explore the transcriptional role of ERα in breast cancer, we performed large-scale analyses of microRNA in 24 hours estrogen treated ERα-expressing T47D cells, Paper II. However, we found no evidence of direct and rapid regulation of mature miRNAs by ERα. In Paper III, we studied ERβ gene regulation in colon cancer cells. We could show that ERβ-expressing xenografts grew significantly slower than those lacking ERβ. Further we demonstrated that ERβ induced a transcriptional response independently of ERα and induced inhibition of the proto-oncogene MYC and other G1-phase cell cycle genes. In Paper IV, we dissected the regulatory networks of ERβ-induced transcriptional changes in human colon cancer cells. The set of genes changed by ERβ varied in different colon cancer cell lines, however, corresponded to the same biological processes such as cell cycle regulation and kinase activity. In addition, we identified the ERβ-driven downregulation of the transcription factor PROX1 as a key mechanism behind a large proportion of the transcriptional changes. In Paper V, we studied the effect of long term expression of ERβ on the miRNA pool in SW480 colon cancer cells. While we could not show a direct and rapid effect of ERα on the miRNome, we showed that long term expression of ERβ did induce large changes in the miRNA pool in colon cancer cells. In particular, we found the oncogenic miR-17-92 cluster to be downregulated and proposed this to be a consequence of the ERβ-induced downregulation of MYC. In conclusion, we have shown that ERβ is antiproliferative in breast and colon cancer cells, both when co-expressed with ERα and alone, as well as identified key signaling pathways. We suggest that activation of ERβ will have a beneficial effect for treatment or prevention of estrogen dependent cancers

Untersuchungen zur Expression der microRNA-Gen-Cluster C19MC und miR-371-3 in Normalgeweben und Tumoren

The miRNA gene clusters C19MC and miR-371-3 are deregulated in a number of tumour entities. In this thesis the expression and regulation of the clusters was examined in thyroid carcinomas, testicular germ cell tumours and placenta. It was demonstrated that both clusters are not activated in thyroid carcinomas and that in testicular carcinomas, C19MC is significantly higher expressed in non-seminomas and tumours in advanced clinical stages. Moreover, C19MC has potential as serum tumour marker for testicular cancer. It was shown that placental mesenchymal stromal cells (MSCs) and chorioamniotic membrane highly express C19MC. Since placental and chorioamniotic mesenchymal stromal are immunosuppressive C19MC miRNAs might play a role in inducing maternal immune tolerance against the placenta and fetus. In case of an immunosuppressive function of C19MC its miRNAs might be applied to treat autoimmune diseases or to suppress the immune response against allogeneic tissue transplants

Epigenetic Alterations in Parathyroid Cancers

Parathyroid cancers (PCas) are rare malignancies representing approximately 0.005% of all cancers. PCas are a rare cause of primary hyperparathyroidism, which is the third most common endocrine disease, mainly related to parathyroid benign tumors. About 90% of PCas are hormonally active hypersecreting parathormone (PTH); consequently patients present with complications of severe hypercalcemia. Pre-operative diagnosis is often difficult due to clinical features shared with benign parathyroid lesions. Surgery provides the current best chance of cure, though persistent or recurrent disease occurs in about 50% of patients with PCas. Somatic inactivating mutations of CDC73/HRPT2 gene, encoding parafibromin, are the most frequent genetic anomalies occurring in PCas. Recently, the aberrant DNA methylation signature and microRNA expression profile have been identified in PCas, providing evidence that parathyroid malignancies are distinct entities from parathyroid benign lesions, showing an epigenetic signature resembling some embryonic aspects. The present paper reviews data about epigenetic alterations in PCas, up to now limited to DNA methylation, chromatin regulators and microRNA profile

Virus-Host Interactions at the Maternal-Fetal Interface

Strategies to protect against viral infections are essential during pregnancy. Maternal-fetal transmission can have serious pathological outcomes, including fetal infection, growth restriction, birth defects, and/or death. Throughout pregnancy, the placenta (composed of polarized trophoblasts amid stromal and vascular arrangements) is an indispensable tissue that forms a barrier at the maternal-fetal interface. Viruses have likely evolved specific mechanisms to exploit the protective functions of placental trophoblasts to initiate fetal infection. Despite the severity of pathologic disease associated with fetal viral infection, little is known regarding virus-host interactions at the maternal-fetal interface. In this work, we have examined the mechanisms by which – 1) placental trophoblasts protect against invading viruses and 2) coxsackievirus B (CVB), a virus associated with fetal pathology, gains entry into polarized trophoblasts. As a model, we have used cultured primary human trophoblasts (PHTs) and immortalized human (BeWo) trophoblasts. We have found that PHTs are highly resistant to infection by six disparate viruses. PHTs transfer this resistance to non-placental recipient cells through exosome-mediated delivery of select placental microRNAs (miRNAs). We show that members of the chromosome 19 miRNA cluster (C19MC), which are almost exclusively expressed in the primate placenta, are packaged within trophoblast-derived exosomes, and attenuate viral replication in recipient cells by inducing autophagy. To study CVB entry into placental trophoblasts, we have merged virological and cell biological techniques, combined with pharmacological inhibitors and siRNAs directed against diverse cellular endocytic and signaling components, to characterize the pathways hijacked by CVB to promote its entry into human trophoblasts. We found the kinetics of CVB entry and uncoating in placental trophoblasts similar to those described in polarized intestinal epithelial cells. CVB entry into placental trophoblasts requires decay accelerating factor (DAF) binding, and is associated with the relocalization of virus from the apical surface to intercellular tight junctions. We have identified a divergent mechanism for CVB entry that is independent of clathrin, caveolae, and dynamin II but is dependent on lipid-rafts and Src family tyrosine kinase signaling. Our studies model viral transmission and infection at the maternal-fetal interface, and have the therapeutic potential for preventing prenatal infections, pre-term labor, and birth defects

Characterization of cellular signalling pathways involved in the regulation of trophoblast cell functions

This publication based doctoral thesis comprises six manuscripts covering the intracellular signaling pathways activated by LIF and OSM and the functional analysis of miR-21 in trophoblastic cells. The aim of this thesis was to elucidate the signaling pathways activated by the cytokines LIF and OSM as well as the role of specific miRNAs in the control of trophoblast cell functions. The results demonstrate that LIF and OSM have similar functions in activating STAT3 and ERK1/2 by changing their phosphorylation status in four trophoblastic cell lines. However, LIF but not OSM significantly increases invasion of trophoblastic cell lines. Inhibition of STAT3 decreases the invasiveness of trophoblastic cell lines by inhibiting the secretion of matrix metalloproteinases (MMPs). These results suggest that LIF shares STAT3 and ERK1/2 activation with OSM but their resulting responses are cell type dependent. The second part of this dissertation is focused on placenta-specific miRNAs and their function on trophoblast cells. The miRNA profile of primary cells includes the expression of at three clusters which are specifically expressed in placenta tissue: C19MC, C14MC and the miR-371-3 cluster. C19MC is highly expressed in third trimester trophoblast cells and in choriocarcinoma cell lines. C14MC is highest expressed in first trimester trophoblast cells as well as in HTR-8/SVneo cells and its plasma concentration decreases with the gestational age. Both miRNA clusters are evolutionary related to the appearance of placenta and their dysregulation can be associated with pregnancy pathologies. In conclusion, this work describes the intercellular signaling pathways activated by OSM and LIF, as well as the function of miR-21 in trophoblastic cells. Moreover, it may provide novel information on their involvement in the pathogenesis of pregnancy-related disorders which may be useful for the design of future non-invasive diagnostic tools or for the development of new therapeutic approaches

Understanding the oncogenic effects of C19MC on hepatocellular carcinoma through epigenetic manipulations.

Aberrant epigenetic alterations, such as DNA methylation, histone modification and miRNA-mediated processes, are associated with several types of cancer including hepatocellular carcinoma (HCC). HCC is the third leading cause of cancer-related fatalities worldwide. Despite the improvements in surgical and medical treatment HCC associated deaths are still showing an increase. Methylation defects at the chromosome 19 miRNA cluster (C19MC) have been shown to be a molecular alteration specific to liver cancers and is an attractive candidate for novel HCC therapies. Several C19MC miRNAs have been reported to be over-expressed in HCC and C19MC hypomethylation may account for this cancer-associated expression. This present study assesses the oncogenic effects of C19MC cluster in HCC using epigenetic manipulations. Using pharmaceutical and novel targeted epigenome editing tools demethylation was induced in HCC cell lines showing a normal hypermethylated state. Demethylation was shown to be sufficient to re-activate C19MC miRNAs throughout the cluster. Following overexpressing miR-512-3p through miRNA mimics, we showed that upregulation of miR-512-3p significantly promotes cell invasion. Since abnormal miRNA expression has been associated with metastatic spread of tumors, studying changes in miRNA expression could help to improve diagnosis and prognosis and provide molecular targets for new therapeutic strategies against HCC. Our study suggested that miR-512-3p can be a robust marker for HCC prognosis and diagnosi