Modulation of Inflammatory Pathways by Glucocorticoids

Glucocorticoids (GCs) are essential steroid hormones widely used as potent anti-inflammatory drugs; however, their clinical use is often accompanied by adverse side effects. The anti-inflammatory action of GCs is exerted through the glucocorticoid receptor (GR) in part by antagonizing the pro-inflammatory nuclear factor kB (NF-kB) whereas the majority of side effects are assumed to be mediated by transactivation of GR target genes. We previously reported the first genome-wide map of GR and NF-kB crosstalk and substantially contributed to the understanding of the networks underlying the glucocorticoid and inflammatory signaling pathways (1).

 

Development of Selective Glucocorticoid Receptor Agonists

Development of selective GR agonists (SEGRA) that will preferentially favor transrepression of pro-inflammatory NF-kB target genes over transactivation of genes associated with undesirable effects is expected to improve the therapeutic potential of GCs. We recently identified two 1,3-benzothiazole analogues as genuine SEGRA using a multidisciplinary approach in collaboration with the Molecular Analysis, Organic and Organometallic Chemistry and Medicinal Chemistry groups of ICB (2).

This research direction aims at developing novel SEGRA and analyzing the molecular mechanism of their action using genome-wide approaches. We use RNA-sequencing to identify the global transcriptome profile of lead SEGRAs and ChIP-sequencing to analyze their effect on the GR and NFKB crosstalk. These studies are expected to reveal signaling pathways underlying the function of SEGRA and to contribute to the continued refinement and improvement of knowledge on the therapeutic potential of SEGRA.

Supported by the following grants

1. STHENOS β: ‘Targeted therapeutic approaches against degenerative diseases with special focus on cancer and ageing-optimisation of the targeted bioactive molecules’, 2017-2021. Program KRIPIS ΙΙ (ESPA 2014-2020), MIS: 5002398.
2. STHENOS: ‘Targeted Therapeutic approaches against degenerative Diseases, with emphasis on cancer and aging’, 2013-2015. Program KRIPIS (ESPA 2007-2013), MIS: 447985.

Selected relevant publications

1. Rao NA, McCalman MT, Moulos P, Francoijs KJ, Chatziioannou A, Kolisis FN, Alexis MN, Mitsiou DJ*, Stunnenberg HG*. Coactivation of GR and NFKB alters the repertoire of their binding sites and target genes. Genome Res. 2011, 21(9):1404-1416.
   doi: 10.1101/gr.118042.110
2. Potamitis C, Siakouli D, Papavasileiou KD, Boulaka A, Ganou V, Roussaki M, Calogeropoulou T, Zoumpoulakis P, Alexis MN, Zervou M*, Mitsiou DJ*. Discovery of New Non-steroidal Selective Glucocorticoid Receptor Agonists. J Steroid Biochem Mol Biol 2019, 186:142-153.
   doi: 10.1016/j.jsbmb.2018.10.007

Depending on the level of expression of estrogen receptor alpha (ERα), progesterone receptor (PR) and HER2 oncogene, breast cancer (BC) is classified as ERα-positive (ERα+/PR+/HER2±), HER2-overexpressing (ERα-/PR-/HER2+) or triple-negative (ERα-/PR-/HER2-). ERα is a marker of BC response to adjuvant endocrine therapy (AET) and favorable disease prognosis. The prognostic and therapeutic significance of ERβ, the second ER isotype, is still open to debate. There are five isoforms of ERβ (ERβ1–5), of which only ERβ1 binds estrogen and antiestrogens. ERβ1 and ERβ2 are the isoforms most frequently expressed in BC. We have developed monoclonal and polyclonal antibodies to ERα, ERβ1 and ERβ2 (1) and used them to show that, i) ERβ2 is a marker of recurrence of ERα-negative BC following adjuvant chemotherapy (2), ii) ERβ2 may have an oncosuppressive role in mammary carcinogenesis (3), and, iii) low ERβ1 and high ERβ2 mark early and late relapse, respectively, of ERα-positive BC following AET (4).  Our ongoing research on the role of ER in BC prognosis and treatment comprises the following research projects:  

Prognostic significance of estrogen receptor beta in early stage ERα-positive breast cancer

Early stage ERα-positive BC is known to recur many years after the standard 5-year-AET, thus increasing the challenge of choosing the optimal adjuvant treatment so as to avoid tumor recurrence. In an effort to unravel the role of ERβ1 and ERβ2 in recurrence risk assessment of early stage ERα-positive BC, we generated clones of MCF7 cells (a model of ERα-positive BC) expressing ERβ1 or ERβ2 and showed that they display high and low sensitivity, respectively, to the antiestrogen tamoxifen (Nolvadex^®). Currently, we are using microarray analysis to identify gene signatures associated with the role of ERβ1 and ERβ2 in modulating the tamoxifen response of the clones. In addition, we are using IHC to assess ERβ1 and ERβ2 expression in formalin-fixed, paraffin-embedded specimens of AET-treated early stage ERα-positive BC in conjunction with specimen RNA-sequencing to derive ERβ1 or ERβ2-associated gene signatures that are potentially able to provide recurrence risk assessments for early stage ERα-positive BC.

Prognostic significance of estrogen receptor beta in triple negative breast cancer

Triple-negative BC (TNBC) represents approx. 15% of all BC cases, expresses Estrogen-Related Receptor α (ERRα) and Epidermal Growth Factor Receptor (EGFR) as well as ERβ1 and/or ERβ2 and, displays high molecular heterogeneity and aggressive clinical behavior. Targeted therapies for the different histopathological categories of TNBC are not available and chemotherapy remains the only treatment option. ERβ1 and ERβ2 have been associated with favorable and unfavorable, respectively, TNBC prognosis, although not in all studies. The aim of this project is to, i) assess the clinical significance of ERβ1 and ERβ2 in TNBC and, ii) use gene expression profiling of TNBC cells subjected to combinatorial targeting of ERβ and ERRα or EGFR in order to discover effective drug combinations that could challenge the viability of these cells.

Understanding the molecular determinants of breast cancer resistance to antiestrogens

The majority of tamoxifen-treated ERα-positive BC cases eventually progresses to tamoxifen-resistance. Aberrant activation of PI3K pathway due to oncogenic mutations of PIK3CA is considered as a major cause of resistance of ERα-positive BC to adjuvant endocrine therapy. The aim of this project is to use gene expression profiling of combinatorial targeting of estrogen receptor and PI3K pathways in BC cells bearing PI3K pathway mutations in order to look for combinatorial treatment options that could overcome resistance of these cells to antiestrogens.

 

Supported by the following grants

• ERBETA: ‘Validation - Reinforcement of the Prognostic Significance of Estrogen Receptor beta in Early Breast Cancer’, 2020-2022. Operational Program ‘Human Resources Development, Education and Lifelong Learning 2014-2020’, MIS: 5050141
• TNBC: ‘Molecular sub-typing of Triple Negative Breast Cancer’, 2012-2015. Program THALES, Grant: 85355.
• POM: ‘PIK3CA Oncogenic Mutations in Breast and Colon Cancers: Development of Targeted Anticancer Drugs and Diagnostics’, 2010-2015. Program COOPERATION, Grant: 09ΣΥΝ-11-675.

Selected relevant publications

1. Chantzi NI, Meligova AK, Dhimolea E, Petrou CC, Mitsiou DJ, Magafa V, Pechtelidou A, Florentin I, Kitraki E, Cordopatis P, Tiniakos DG, Alexis MN*. Insights into ectopic estrogen receptor expression, nucleocytoplasmic distribution and interaction with chromatin obtained with new antibodies to estrogen receptor α and β. Steroids 2011, 76(10-11):974-985.
   doi: 10.1016/j.steroids.2011.05.010
2. Chantzi NI, Tiniakos DG, Palaiologou M, Goutas N, Filippidis T, Vassilaros SD, Dhimolea E, Mitsiou DJ, Alexis MN*. Estrogen receptor beta 2 is associated with poor prognosis in estrogen receptor alpha-negative breast carcinoma. J Cancer Res Clin Oncol. 2013, 139(9):1489-1498.
   doi: 10.1007/s00432-013-1467-4
3. Chantzi NI, Palaiologou M, Stylianidou A, Goutas N, Vassilaros S, Kourea HP, Dhimolea E, Mitsiou DJ, Tiniakos DG, Alexis MN*. Estrogen receptor β2 is inversely correlated with Ki-67 in hyperplastic and noninvasive neoplastic breast lesions. J Cancer Res Clin Oncol. 2014, 140(6):1057-1066.
   doi: 10.1007/s00432-014-1652-0
4. Dhimolea E, Tiniakos DG, Chantzi ΝΙ, Goutas N, Vassilaros SD, Mitsiou DJ, Alexis ΜN*. Estrogen receptors β1 and β2 are associated with distinct responses of estrogen receptor α-positive breast carcinoma to adjuvant endocrine therapy. Cancer Lett. 2015, 358(1):37-42.
   doi: 10.1016/j.canlet.2014.12.022

 

Development of raloxifene analogues of high endometrial safety

Menopausal decline in estrogen levels is associated with vasomotor symptoms, the genitourinary syndrome of menopause (GSM), osteoporosis, senile dementia, and several other disorders. Hormone replacement therapy (HRT) is known to prevent menopausal disorders but increases uterine cancer risk. The Selective ER Modulator (SERM) raloxifene (Evista®) is prescribed for osteoporosis prevention and treatment.  However, long term compliance with Evista® is low, partly because raloxifene cannot relieve vasomotor symptoms and its stimulation of the uterus is not negligible. We recently reported that raloxifene analogues with a bulky basic side chain amino group could potentially provide for higher endometrial safety treatment of the menopausal syndrome (1). A new series of raloxifene analogues is presently available to be tested for endometrial safety in the absence and presence of low systemic estrogen.  

Natural products with osteoprotective and/or GSM-preventive potential

Given that systemic estrogen increases the risk for endometrial adenocarcinoma, post-menopausal women tend to prefer herbal remedies to systemic or topical estrogen. In this light we evaluated hundreds of plant extracts and isolated compounds for estrogen-like and osteoblasto-/osteoclasto-genic activity using reporter cell lines and tested selected compounds and extracts for osteoporosis prevention and endometrial safety in the ovariectomized (OVX) rat model of estrogen deficiency. We reported, among other findings, i) on natural compounds that stimulate ER-mediated gene expression but not cancer cell proliferation and hence might act as cancer chemopreventive agents (2); ii) on isoflavonoid-rich extracts that display high vaginotrophic vs weak uterotrophic activity in the OVX rat and thus might safely substitute for low-dose vaginal estrogen in treating GSM (3); iii) that the ex-vivo estrogen-like, osteoblastic and anti-inflammatory activities of genistein glucosides exceed those of the aglycone, which is advantageous efficacy-wise considering the much higher abundance of glucosides compared to aglycones in plant-derived extracts (4); and iv) that preparations rich in some Genista extracts could potentially substitute for low-dose vaginal estrogen often prescribed for the GSM (5). More natural and nature-derived products are on the way to be assessed ex-vivo and in the OVX mouse model as potential chemopreventive agents for osteoporosis and/or the GSM. 

Supported by the following grants

1. SERMENCO: ‘Development of new Selective Estrogen Receptor Modulators for preventing mENopause Consequences’, 2012-2015. Program THALES, Grant: 80038.
2. OSTEOPRO: ‘Development of natural products and analogues for OSTEOporosis PReventiΟn: transcriptomic, proteomic and metabolomic approaches’, 2011-2015. Program COOPERATION, Grant: 09ΣΥΝ-11-1076

Selected relevant publications

1. Lambrinidis G*, Gouedard C, Stasinopoulou S, Angelopoulou A, Ganou V, Meligova AK, Mitsiou DJ, Marakos P, Pouli N, Mikros E*, Alexis MN*. Design, synthesis, and biological evaluation of new raloxifene analogues of improved antagonist activity and endometrial safety. Bioorg Chem. 2021 Jan;106:104482.
   doi: 10.1016/j.bioorg.2020.104482
2. Polasek J, Queiroz EF*, Marcourt L, Meligova AK, Halabalaki M, Skaltsounis AL, Alexis MN*, Prajogo B, Wolfender JL, Hostettmann K. Peltogynoids and 2-phenoxychromones from Peltophorum pterocarpum and evaluation of their estrogenic activity. Planta Med. 2013 Apr;79(6):480-6.
   doi: 10.1055/s-0032-1328299
3. Tchoumtchoua J, Makropoulou M, Ateba SB, Boulaka A, Halabalaki M, Lambrinidis G, Meligova AK, Mbanya JC, Mikros E, Skaltsounis AL, Mitsiou DJ, Njamen D, Alexis MN*. Estrogenic activity of isoflavonoids from the stem bark of the tropical tree Amphimas pterocarpoides, a source of traditional medicines. J Steroid Biochem Mol Biol. 2016, 158:138-148.
   doi: 10.1016/j.jsbmb.2015.12.015
4. Fokialakis N*, Alexi X, Aligiannis N, Boulaka A, Meligova AK, Lambrinidis G, Kalpoutzakis E, Pratsinis H, Cheilari A, Mitsiou DJ, Mitakou S, Alexis MN*. Biological evaluation of isoflavonoids from Genista halacsyi using estrogen-target cells: Activities of glucosides compared to aglycones. PLoS One. 2019 Jan 8;14(1):e0210247.
   doi: 10.1371/journal.pone.0210247
5. Cheilari A, Vontzalidou A, Makropoulou M, Meligova AK, Fokialakis N, Mitakou S, Alexis MN*, Aligiannis N*. Isoflavonoid Profiling and Estrogen-Like Activity of Four Genista Species from the Greek Flora. Molecules. 2020 Nov 24;25(23):5507.
   doi: 10.3390/molecules25235507

Endocrine-disrupting chemicals are known to interfere with hormone action, to affect homeostasis, reproduction and endocrine gland development, and to potentially increase endocrine cancer risk. We are using cell and animal models to test the endocrine-disrupting potential of environmental estrogens (e.g. Bisphenol A) and androgens (e.g. TBECH, DPTE). Bisphenol A (CID 6623) is found in the plastic packaging of consumer goods, is known to exert estrogenic effects and is thought to increase breast cancer risk. In collaboration with the lab of Prof. E. Kitraki (Athens Univ.) we have shown that chronic perinatal exposure of rats to a low dose of Bisphenol A affects adrenal histology and perturbs the basal and stress-induced activity of hypothalamic–pituitary–adrenal axis, which may increase susceptibility to stress-related disorders in later life (1). The brominated flame retardants TBECH (CID 18728) and DPTE (CID 88314418) are used to reduce the flammability of commercial and industrial products and are readily detectable in the food web and in internal and marine waters. In collaboration with the lab of Prof. P.E. Olsson (Orebro Univ.) we have recently shown that TBECH and DPTE alter prostate growth, histology and gene expression in a manner that may increase prostate cancer risk (2).

 

Relevant publications

1. Bereketoglu C, Modig C, Pradhan A, Andersson PL, Stasinopoulou S, Mitsiou DJ, Alexis MN, Olsson PE. The brominated flame retardants TBECH and DPTE alter prostate growth, histology and gene expression patterns in the mouse. Reprod Toxicol. 2021 2021 Jun;102:43-55.
   doi: 10.1016/j.reprotox.2021.04.002
2. Panagiotidou E, Zerva S, Mitsiou DJ, Alexis MN, Kitraki E. Perinatal exposure to low dose bisphenol A affects the neuroendocrine stress response in rats. J Endocrinol. 2014 Jan 27;220(3):207-18.
   doi: 10.1530/JOE-13-0416

MSc Programs

Group members participate (organization/teaching) in the following postgraduate programmes:

•  ’Oncology: from oncogenesis to therapy’, joint Master's Degree co-organized by ICB/NHRF and the Medical School, University of Crete, Greece (2019-2022). http://www.english.med.uoc.gr/oncology
• ‘Bioentrepreneurship’, joint Master's Degree co-organized by ICB/NHRF and the University of Thessaly, Greece (2015-2022).
  http://bioepixirin.bio.uth.gr/
• ‘Molecular & Applied Physiology’, Master's Degree organized by the Medical School of the National & Kapodistrian University of Athens (2018). https://www.molecularandappliedphysiologymedicaluoa.com/

PhD Thesis

• Dimitra Siakouli: ‘Development of selective non-steroidal glucocorticoid receptor agonists and evaluation of their anticancer activity’, Medical School, National & Kapodistrian University of Athens (full supervision, ongoing).
• Argyro Vontzalidou: ‘Development of natural products and their synthetic analogues in order to treat menopausal disorders and to evaluate their effect on the activity of the enzyme tyrosinase’, Faculty of Pharmacy, National & Kapodistrian University of Athens (partial supervision, 2018)
  https://pergamos.lib.uoa.gr/uoa/dl/object/uoadl:2837985

MSc Thesis

• Despoina S. Xenopoulou: ‘The role of estrogen receptor beta in the response of early breast cancer to antiestrogens and retinoids’, Joint Master’s Degree ‘Oncology: from Oncogenesis to Therapy’, University of Crete/National Hellenic Research Foundation (ongoing).
• Eleni Florou: ‘Specificity assessment and use optimization of new antibodies against estrogen receptors alpha and beta’, MSc ‘Neoplastic Disease in Humans’, Medical School, National & Kapodistrian University of Athens (ongoing).

Undergraduate Thesis

• Maria Zoumpouli: ‘Prognostic significance of estrogen receptor beta in early breast cancer’, Department of Biotechnology, Agricultural University of Athens (ongoing)
• George Panagiotou: ‘Development of selective non-steroidal glucocorticoid receptor agonists: biological evaluation of new compounds’, Department of Biology, National & Kapodistrian University of Athens (ongoing)

Training Internships

Eleftheria Panagiotopoulou (2022), Myrto Charouli (2019), Chrysanthi Tsirligkani (2019), Elza Chotza (2018), Sotiria Skiani (2018), Maria-Eleni Pouliou (2018).

 

Plan A: Cancer patient-centered precision medicine technologies

Breast cancer (BC) is the most common cancer among women. While BC incidence rate keeps increasing, BC mortality rate has dropped by 39% since the late 1980s mostly due to early diagnosis and targeted therapies. The Molecular Endocrinology (ME) Group participates in the proposal “Cancer patient-centered precision medicine technologies, new drivers for transforming the RTDI ecosystem in Cyprus and Greece”, submitted in March 2022 in response to the call “HORIZON-WIDERA-2022-ACCESS-04 (Excellence Hubs)”. The project aims at using precision medicine technologies such as genomics of 3D organoids to determine a particular woman’s frequency of monitoring and sampling, so as to secure early diagnosis and patient-tailored targeted treatment. Participation of the Institute of Chemical Biology in the Hellenic Network for Precision Medicine on Cancer (https://oncopmnet.gr/) will help support the objectives of the project (in collaboration with 3 other ICB groups and 19 other public and private entities in Greece and Cyprus).

 

Plan B: Prognostic significance of estrogen receptor beta isoforms ERβ1 and ERβ2 in early stage breast cancer

The ERα-positive breast carcinoma (ERα+ BC) constitutes more than 70% of all BC cases. Adjuvant endocrine therapy (AET) with antiestrogens or aromatase inhibitors is the treatment of choice for early stage ERα+ BC. This is known to recur even many years after completion of the standard 5-year-AET. Specific prognostic tools able to accurately assess recurrence risk are therefore of great importance. The role of low ERβ1 and high ERβ2 ERβ isoforms as markers of early and late recurrence risk (Dhimolea et al, 2015, doi: 10.1016/j.canlet.2014.12.022) will be assessed through isoform-specific gene signatures obtained from ERβ1- and ERβ2-expressing BC cells and from FFPE specimens of early and late recurrent AET-treated ERα+ BC using RNA-sequencing (in collaboration with the Dept. of Pathology, Aretaieion Hospital, Medical School, Univ. of Athens).

Plan A: Development of innovative food supplements from Greek medicinal plants for preventing postmenopausal osteoporosis

Osteoporosis is one of the most serious consequences of menopause. It is associated with increased morbidity and mortality and its management is imperative for improving quality of life and for reducing its financial burden on the health system. An increase in pro-inflammatory molecules due to low menopausal estrogen levels contributes to the development of osteoporosis.  Selected extracts from Greek flora will be evaluated for their osteoprotective, estrogenic and anti-inflammatory activities using an analytical platform to detect bioactive ingredients and a series of cell-based assays as well as animal models to identify promising osteoprotective agents. Development of natural products for the prevention of postmenopausal osteoporosis responds to the needs of the market and to the perception of modern society on the chemopreventive potential of natural products (in collaboration with the Dept. of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, Univ. of Athens and a private sector entity).

 

Plan B: Development of innovative products from edible Greek plants for enhancing memory and learning

About 47 million people suffer today from dementia and this number is expected to double by 2040. The initial stage of dementia, known as mild cognitive impairment (MCI), is a noticeable and measurable decrease in cognitive abilities which goes beyond the impairment associated with normal aging. Cholinergic dysfunction, oxidative stress and inflammation play important roles in inducing and promoting memory decline and MCI. There is growing tendency among the adult population to enhance cognitive function using brain health supplements. Preparations from endemic non-toxic Greek plants, known for their contribution in improving memory, sleep quality and mood will be tested in hippocampal neuronal cells, in mice rendered amnestic following scopolamine administration and in people with MCI for prevention of memory decline (in collaboration with the Dept. of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, Univ. of Athens and a private sector entity). 

Plan Α: Development of Selective Glucocorticoid Receptor Agonists (SEGRA) and evaluation their anti-inflammatory activity and their mechanisms of action using genome-wide approaches

Glucocorticoids (GCs) are used as potent anti-inflammatory drugs predominantly through transrepression of NFKB target genes; however, GC therapy is often accompanied by adverse side effects predominantly due to transactivation of GRE-dependent genes. We intend to, 1) apply a ‘hit-to-lead’ optimization approach to develop highly selective SEGRA from hit compounds using in silico docking in combination with Molecular Dynamics simulations for hit-to-lead optimization, 2) subsect the best lead compound to in vivo evaluation of anti-inflammatory action and, 3) analyze its mechanisms of action in target cells using genome-wide approaches (in collaboration with other ICB groups).

 

Plan B: Evaluation of the anti-inflammatory activity of natural compounds

We evaluate the anti-inflammatory activity of plant extracts and isolated compounds using i) reporter cell lines (stably transfected with an NF-kB reporter gene) treated with either lipopolysaccharide (LPS) or tumor necrosis factor (TNF) to elicit an inflammatory response, and ii) a zymosan-induced paw edema in mice (in collaboration with the Dept. of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, Univ. of Athens and a private sector entity).