Giving Another Chance to Mifepristone in Pharmacotherapy for Aggressive Meningiomas—A Likely Synergism with Hydroxyurea?

Curr Probl Cancer 40 (2016) 229–243

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Curr Probl Cancer

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Giving another chance to mifepristone in pharmacotherapy for aggressive meningiomas—A likely synergism with hydroxyurea?

İlhan Elmaci, MDa, Meric A. Altinoz, MDb,*, Aydin Sav, MDc, Zeliha Yazici, PhDd, Aysel Ozpinar, PhDe

Introduction

Meningiomas: An underestimated health problem

Meningiomas are the most frequently reported intracranial tumors, accounting for approximately one-fourth of all reported primary brain neoplasms.1 They are benign in approximately 90% of the cases and the remaining cases are either borderline or atypical (World Health Organization [WHO] grade II) or malignant (WHO grade III).1 In the United States, the incidence rates with similar age standardization estimated from ﬁgures provided by the Central Brain Tumor Registrywere1.8formenand4.2per100,000forwomenin2006.1 Increasing incidence rates of meningiomas have been reported from several industrialized countries since the early 1980s. But recent studies revealed that real meningioma incidence is even higher.1 As meningiomas are mostly benign, they are not covered by most cancer registries.1 Nevertheless, in Finland, as in other Nordic countries, all surgeons and pathologists are obliged to report all tumors of the central nervous system, both malignant and benign, to the cancer registry.1 To reveal the real incidence of

Grant/Financial Support: none. This is original work and is not under consideration for publication elsewhere. This submission is for the special issue being compiled by Beata Holkova on the topic Amylodosis. a Department of Neurosurgery, Memorial Hospital, Istanbul, Turkey b Department of Immunology, Experimental Medical Research Institute/DETAE, Istanbul University, Istanbul, Turkey c Nisantasi Neuropathology Group, Istanbul, Turkey d Department of Pharmacology, Cerrahpasa Faculty of Medicine, Istanbul University, Turkey e Department of Biochemistry, Acibadem University, Istanbul, Turkey * Corresponding author: Meric A. Altinoz, MD, Guven Sk, Kagithane, Yildirim Apt. No: 5 D: 6, İstanbul. Tel.: +90 536 201 8327. E-mail address: [email protected] (M.A. Altinoz). http://dx.doi.org/10.1016/j.currproblcancer.2016.05.001 0147-0272/& 2016 Elsevier Inc. All rights reserved. 230 İ. Elmaci et al. / Curr Probl Cancer 40 (2016) 229–243 meningiomas, a comprehensive material was investigated by compiling hospital sources with the Finnish Cancer Registry database.1 The corrected age-standardized meningioma incidence rate was 2.9 per 100,000 for men and 13.0 per 100,000 for women, a third higher than the cancer registry ﬁgures.1 The researchers underlined that even these incidences may be lower than the real values, as only operated cases were included to the study. Furthermore, there exist studies that showed incidental meningiomas in 1% of asymptomatic volunteers.1 If we exclude incidental cases or asymptomatic meningiomas being followed clinically, a projection based on Finnish results would reveal that approximately 51,400 patients with meningioma are operated in the United States each year including 5140 patients having atypical or malignant tumors. High-grade meningiomas frequently recur despite surgery and radiotherapy and are therefore associated with poor overall survival.2 Anaplastic meningiomas (WHO grade III, 2007 WHO Classiﬁcation) are particularly aggressive with a median overall survival time of 15 months.3 At present, guidelines recognize the following 3 medical therapies for inoperable and radiation-refractory meningiomas: hydroxyurea (HU), interferon alfa, and Sandostatin LAR, a somatostatin analogue.4 As would be discussed below, targeting angiogenesis is also suggested as a novel approach in the management of high-grade meningioma. But before that, we would give a short description regarding the sex difference and complex endocrinology of meningiomas.

Sex difference and endocrinological complexity of meningiomas

As would be discussed in detail, pregnancy is an accelerating factor for the growth of meningiomas, yet nonpregnant women also develop meningiomas with higher incidences than men. The exact mechanism of this difference is unknown. However, there exist some clues. Clinical complaints of women patients with meningioma may also increase during menstruation, and several authors have reported an association between meningioma and breast cancer.5 Therefore, estrogen may be blamed as a stimulating factor for meningioma growth and as the responsible factor for sex difference. Indeed, regression of meningiomas with antiestrogen mepitiostane has been reported.5 But here, one should also admit that some investigators showed that 40% of meningiomas also express androgen receptor, and androgen receptor antagonists may also slower meningioma growth.5 Some groups also suggested that progesterone and androgen receptor expression in meningiomas did not change with sex and hence sex steroid receptor expression does not associate with the sex difference.5 So what could be the exact reason for the women preponderance in meningiomas? When sex steroid receptors including estrogen, androgen, and progesterone receptors (PRs) were compared with Ki-67 proliferation index (PI) in 443 meningioma samples, it was revealed that only the estrogen receptor correlated with cell proliferationinboththesexes.5 Furthermore, it shall also be kept in mind that the rate of the androgen receptor expression in whole meningiomas is about the half of PRs.5 Cytosolic receptors of the sex steroids belong to the steroid-thyroid receptor superfamily, and these receptors are not 100% speciﬁc for each sex steroid. Hence, it is very likely that not a single sex steroid receptor, rather the sum effects of steroid receptors and the ratio of their ligands modify the meningioma cell response to sex steroids and determine the sex difference. As estrogen receptor is the predominant proliferating receptor among other sex steroid receptors, a high estrogen-to- androgen ratio may explain a strong stimulus of estrogen in women. On the contrary, despite androgen receptors may also act as proliferative agents, they are expressed approximately half the ratios of PRs, and estrogen receptors are not additionally saturated with estrogen in men.5 As would be mentioned below, such a sum effect may also explain why the PR expression does not highly correlate with the meningioma response to the PR antagonist, mifepristone.

Higher angiogenesis in high-grade meningiomas

Microvessel density (MVD) correlates with enhanced tumor cell proliferation and tumor grade in human meningioma.6 Vascular endothelial growth factor (VEGF) is highly produced in İ. Elmaci et al. / Curr Probl Cancer 40 (2016) 229–243 231

Fig. A schematic representation of antitumor pathways activated by mifepristone and HU. The possibility of likely synergisms at multiple levels was suggested. (Color version of ﬁgure is available online.) malignant meningiomas, and a signiﬁcant regression of a recurring anaplastic meningioma was reported following bevacizumab (VEGF-A antagonist) treatment.5 VEGF receptor 2 messenger ribonucleic acid exists in 75% of recurring meningiomas vs none of those which did not recur (P ¼ 0.007).7 MVD and PI are positively correlated and time to recurrence is also shorter in patients with high MVD (P ¼ 0.027).7 Treatment with bevacizumab in 15 patients with atypical (WHO grade II) or anaplastic (WHO grade III) meningioma was well tolerated with a median progression-free survival (PFS) of 26 weeks and with a 6-month PFS rate of 43.8%.8 Hence, in patients who have exhausted radiation and surgical options, antiangiogenic drugs may be considered including HU and mifepristone, as both exert antiangiogenic activities (Fig).

Role of hydroxyurea in recurring and high-grade meningiomas

The HU blocks DNA synthesis by decreasing deoxyribonucleotide production via inhibiting the R2 subunit of ribonucleotide reductase (RRM2).9 Ribonucleotide reductase, consisting of 2 subunits (RRM1 and RRM2), is a rate-limiting enzyme in deoxynucleotide production for DNA synthesis and it plays an important role in cell proliferation and tumorigenicity. Reduction of intracellular deoxyribonucleotides also block DNA excision repair,10 leading to tumor radiosensitization with HU.9 Despite being synthesized very early in 1869, HU is still in use for the treatment of myeloproliferative diseases and chronic myeloid leukemia.10,11 HU is generally administered orally with excellent absorption from the gastrointestinal tract and with a very good oral bioavailability ranging from 80%-100%. At tissue level, HU easily enters both brain and cerebrospinal ﬂuid.12 In many studies, the oral dosage given was 20-30 mg/kg/d (1.4-2.1 g/70 kg normal-weight patient) or 1 g/m2/d (average body surface areas of normal women and men are 1.6 and 1.9 m2, respectively, corresponding to approximate doses of between 1.6 and 1.9 g/d).12 HU was ﬁrst tested in 1997 in 4 patients with inoperable meningiomas invading the cavernous sinus. Tumor shrinkage occurred in 3 patients and stabilization occurred in all patients.12 After5yearsofthisstudy,35patientswithWHOgradeII(n ¼ 22) or III (n ¼ 13) meningioma were reported who were treated with HU following disease progression after surgery and radiotherapy.13 HU was given at a dose of 1 g/m2 orally twice a day (3.2- 232 İ. Elmaci et al. / Curr Probl Cancer 40 (2016) 229–243

3.8 g in total) and 1 cycle was defined as 4 weeks of daily HU. Patients received 0.5-7 cycles (median ¼ 2.0) of HU with modest toxicity (28.5% all grades and 8.5% grade 3þ anemia or fatigue).13 Further, 43% of patients had stable disease at first evaluation, yet the overall PFS rate was 3% at 6 months. In this series, HU though well tolerated appeared to have very limited activity for high-grade meningiomas.13 In the same year, results of HU therapy at same doses yet applied to patients with WHO grade I and gradeIImeningiomaswerereported.11 A total of 13 patients with recurrent WHO grade I or II meningioma were treated with HU, and treatment was continued until disease progression or until onset of unmanageable toxicity. Of the 13 patients, 10 patients (77%) showed stable disease, with time to progression with a median of 72.4 months (6 patients were still accruing time at the time of publication).11 However, 3 patients had progressive disease after 88, 89, and 36 months, respectively, and there were no severe (grade III-IV) hematological and systemic side effects.11 HU was modestly active against recurrent meningiomas and induced long-term stabilization of disease in some patients, most likely those with enlarging benign tumors.11 Meningiomas frequently express platelet-derived growth factor receptor (PDGFR)-α and PDGFR-β, whereas a percentage of meningiomas also express PDGF, suggesting autocrine and paracrine growth loops.1 Imatinib (Gleevec) is an inhibitor of PDGFR-α and PDGFR-β, as well as other oncogenic kinases including bcr-abl, c-kit, and c-fms.1 Hence, it was assumed that HU- imatinib in combination may target both growth signals and active DNA synthesis. Thus, an open-label, dual-center, phase II trial was conducted, where daily imatinib and HU were applied to 13 patients with recurrent or progressive meningiomas.1 Imatinib was applied either at 400 or 500 mg twice a day, the latter dose given to patients taking antiepileptic drugs, which metabolize imatinib at higher rates. Of 13 patients, 4 patients achieved stable disease for at least 6 months (range: 7-18 months), suggesting a modest benefit.1 Here, it should be noted that the applied HU dose of 1 g was much less than dosages of 3.2-3.8 g, which were applied for the treatment of meningiomas in other clinical studies.5,11 Hence, further studies are necessary to reveal whether higher dosages of HU may exert a more pronounced synergism with imatinib. Indeed, a veterinary case of meningioma revealed striking activity of imatinib in combination with HU at doses applicable to humans as a single agent.14 Computed tomography imaging revealed a suspected cerebellar meningioma in a dog that was referred because of facial paresis and ataxia.14 HU (50 mg/kg, [corresponding to 3.5 g for a 70-kg weight of human] every other day) and prednisolone 2 mg/kg/d were administered. The mass reduced in size after 4 weeks, yet regrew after 6 weeks. Imatinib (8 mg/kg/d) was then added to HU therapy, which then led to continuous and prominent tumor reduction.14 After the dog died due to anesthesia during one of the follow-ups, postmortem histopathology confirmed a transitional meningioma.14 As both HU15 and imatinib16 trigger apoptosis in malignant cells, it was assumed that the sustained tumor shrinkage may have occurred due to cell death. Lastly in 2014, a case of anaplastic meningioma was reported with a near- complete radiological regression after 5 months of 25 mg/kg/d (equivalent to 1.5 g) of HU therapy.9 HU was given within 1 month after stereotactic radiotherapy, and hence it was speculated that this remarkable response may have occurred via HU interaction with cellular radiotherapy effects.9

Antiangiogenic activity of HU

One of the current clinical applications of HU is essential thrombocythemia (ET), and angiogenesis plays an important role in ET.17 The VEGF levels are significantly higher in patients with untreated ET with respect to controls, and HU therapy reduces VEGF levels.17 Recently, effects of HU (50-200 μM; 17-24 h) on angiogenesis were studied.18 Following HU therapy, human umbilical vein endothelial cells displayed a 75% inhibition of capillarylike structure formation and significant decrease in proliferative and invasive capacities.18 Furthermore, HU significantly decreased angiogenesis-triggering hypoxia inducible factor 1α expression. More- over, HU reduced VEGF-induced vascular development for more than 7 days in vivo.18 Considering high angiogenesis in anaplastic meningiomas and the antiangiogenic actions of mifepristone (outlined below), a strategy to combine mifepristone and HU seems appropriate. İ. Elmaci et al. / Curr Probl Cancer 40 (2016) 229–243 233

Applications of intense doses of HU in clinical oncology

HU has a dose-limiting myelosuppressive toxicity that is generally manageable and reversible, and doses of HU could be escalated to very high levels with significant tumor responses. Nonetheless, this is not widely recognized, and a broader awareness of this fact may widen the applicability of HU in diverse cancers including high-grade meningiomas. In 1988, a series of studies were conducted regarding in vitro and clinical efficacy of ultra-high doses of HU.19 HU, at a level of 1 mM, inhibited growth of a human lung cancer cell line by 99%. In 18 patients with lung cancer, serum levels of more than 1 mM HU were obtained by escalating doses of HU via continuous intravenous (iv) infusion at 3-weekly intervals.19 Huge dose increments from 24 g in 24 hours to 48 g in 48 hours were achieved. The dose-limiting toxicity at 48 g was myelosuppression. A mean serum level of greater than 1 mM was achieved by 6 hours and then maintained during treatment, whereas oral administration of HU did not result in similar sustained levels.19 These findings provided a basis for combination of HU as a DNA repair inhibitor with other chemotherapeutics.19 This would in particular make sense for chemotherapeutics that could pass through the blood-brain barrier and were previously applied for brain tumors, such as carmustine and fotemustine. Indeed, clinical efficacies were demonstrated for HU combinations with carmustine for melanoma20 and with carmustine21 and radiotherapy22 for glial tumors. In addition, despite 1 study to the contrary,23 other studies showed efficacy of HU in combination with imatinib in glial tumors.24,25 Yet, all of these studies that revealed modest benefits were applied with relatively low HU doses. HU exhibits prominent synergism with imatinib in glioma cells in vitro,26 and fotemustine inhibits the HU- target ribonucleotide reductase.27 Hence, escalating HU doses to high levels in combination with these drugs may exert prominent efficacy in future trials. Other clinical studies also exist which applied high doses of HU for various malignancies. For instance, HU was applied at 9 g/d for chronic myeloid leukemia treatment.28 HU at a dose of 1.5 g/ m2 (2.4-2.9 g) was given as an adjunct to multimodal chemotherapy with autologous marrow transplantation, which led to 33% and 24% complete remissions (CRs) in metastatic breast cancer and refractory large cell lymphoma, respectively.29 A phase II study was conducted to treat metastatic melanoma with high-dose HU and dacarbazine (DTIC).10 As dacarbazine (DTIC) induces methylated DNA adducts, it was combined with high-dose HU for further inhibition of DNA repair and to achieve higher tumor cell death.30 A total of 16 patients with metastatic melanoma who did not receive prior therapy were treated with HU as a continuous iv infusion of 1 g/h (total ¼ 36 g) and DTIC (1 g/m2).31 Of the 4 patients, 3 patients had predominant visceral metastases, achieved partial remission with an objective remission rate of 25%. Disease was stabilized in 5 patients with a median time to progression of 16 months. Only 1 patient developed grade 3 leukopenia and septicemia.32 These results show that even in malignancies with very poor prognosis, adjunct high- dose HU may provide significant clinical results. A phase I/II study evaluated the incorporation of HU into high-dose chemotherapy of non-Hodgkin'slymphoma.33 Patients with primary refractory and refractory-relapsed non-Hodgkin's lymphoma were treated with carmustine, cyclophosphamide, etoposide, and HU (BCHE) with autologous hematopoietic stem cell rescue, among whom 21 patients received HU in a dose escalation of 2-12 g/m2 (3.2-3.8 to 19.2-23.2 g) by 72-hour continuous iv infusion.33 When the iv formulation was not available, patients were given 18 g/m2 of HU orally in divided doses every 6 hours over the same period. Further, 45% achieved CR, and more patients treated with iv BCHE achieved CR than patients treated with oral BCHE (57% vs 29.4%). The 11.8% of oral BCHE and 30% of iv BCHE-receiving patients remained disease free with a minimum follow-up time of 3 years.33 Higher response rate with iv BCHE translated into a significantly superior probability of PFS (33% at 4 years vs 12% for oral BCHE).33 Interestingly, continuous iv HU acted both more effective and less toxic than intermittent oral HU in this regimen. Antileukemic effectiveness and toxicity of high-dose HU was reported in the treatment of poor-risk myeloid leukemias.30 Further, 12 adult patients with high-risk acute myeloid leukemia (AML) (4 in early relapse, 7 with secondary AML, and 1 with de novo AML concomitant to a lymphoproliferative disorder) received HU (100 mg/kg/d, approx 7 g/d) until marrow aplasia or for a maximum of 30 days.30 A total of 5 patients (41.6%) achieved CR, 1 achieved partial remission, 4 were resistant, and 2 died from infection. These results were found encouraging, warranting further studies.30 234 İ. Elmaci et al. / Curr Probl Cancer 40 (2016) 229–243

Altogether, high-dose prolonged infusion of HU may provide signiﬁcant responses in refractory malignancies. In the case of meningiomas, before starting with oral HU therapy, high-dose continuous infusions of HU may provide death of clonogenic clones in these tumors, and its concomitant application with mifepristone may uncouple antitumor efﬁcacy vs systemic toxicity.

Meningioma: Effect of pregnancy and progesterone and expression of progesterone receptors

In 1929, Harvey Cushing demonstrated accelerated growth of meningiomas in pregnancy,34 a finding which has been reaffirmed and substantiated in the literature.31 It was proposed that progression of meningiomas in pregnancy is due to angiogenesis and edema upon analysis of 17 patients.31 Researchers witnessed focal CD34þ hemangioma-like hypervascularization in 8 tumors, intracellular or extracellular edema or both in 14 tumors, and bleeding and necrosis in approximately 30% of tumors.31 Angiogenesis and edema may indeed contribute to gestational meningioma progression; yet it should also be kept in mind that edema and necrosis are general features of tumors with high cell proliferation. Moreover, abundant data indicate that progesterone accelerates meningioma growth, as would be discussed here. The expression of PR in meningiomas is reported at different percentages, which may depend both on tumor subtype and differing immunohistochemical methods. More recent studies report higher percentages of PR-positivity, likely because of the application of more sensitive antibodies. A relatively recent study analyzed PR expression both in tumoral and normal meningial cells.35 PR expression rate was determined to be more than 60% in meningiomas, and surprisingly normal meningial cells also expressed PR at a rate of 47%.35 This finding demonstrates that progesterone has as yet an undefined role in biological regulation of meningial cell proliferation and differentiation. Additionally, the same study also demonstrated that tumors in women contained PR at higher percentages than those in men.35 Most epidemiological studies indicate that female sexual hormones stimulate meningioma growth. Menopause and oophorectomy decrease the risk of meningioma, where obesity—which increases estrogen levels—enhances this risk.32 Contraceptives are agents that contain progesterone analogues either alone or in combination with estrogens. It was observed that contraceptives increase the risk of meningiomas with odds ratio changing between 1.39 and 1.5.36 The first in situ evidence that progesterone analogues stimulate meningioma growth was published in 2010.37 In a patient who used the progesterone analogue megestrol acetate for a prolonged period, multiple intracranial meningiomas developed; and following cessation of treatment, all tumors had strikingly regressed and one had completely disappeared.37 Pathologic examination revealed that more than 25% of tumor nuclei stained positive with PR with most of the expressed PR isoforms being PR-B rather than PR-A.37 This finding is similar to that in gliomas. In high-grade human glial tumors, the dominantly expressed PR isoform is PR-B, whereas the level of PR-A exerts an opposite correlation with PI.38,39 Similar to the first case, in a female patient who received multiple sequences of infertility treatment, a giant meningioma had developed and pathologic investigation revealed that the tumor cells were intensely stained with PR.40 Despite the stimulating role of pregnancy and progesterone analogues on growth of meningiomas, there also exists evidence that PR negativity is correlated with aggressive forms of meningioma. When the aforementioned fact is kept in mind, that the PR is expressed at significant rates in normal meningial cells,35 the PR-positivity in low-grade meningiomas can be explained with high differentiation and its loss with enhanced cellular anaplasia. In a study conducted in 2010, PR expression rates were found to be 96.8%, 20%, and 0% in WHO grade I, grade II, and grade III meningiomas, respectively.41 In 2011, 60 meningiomas were analyzed regarding prognosis and tumoral PI, PR, and human epidermal growth factor receptor 2 (HER2) expressions.42 It was observed that the tumors with high HER2 expression were characterized by high PI and low PR expression, and that the mean PI values of PR-negative and HER2-positive tumors were found to be significantly higher than PR-positive and HER2-negative tumors.42 High PI and PR negativity were also found to significantly correlate İ. Elmaci et al. / Curr Probl Cancer 40 (2016) 229–243 235 with tumor recurrence.42 Lastly in 2014, a study conducted on 175 meningiomas revealed PR- positivity on 87% of tumors and confirmed reduction of PR expression in aggressive tumor subtypes.43

Antiproliferative efﬁcacy of mifepristone on meningioma in vitro, in vivo, and in clinical applications

Antiproliferative efficacy of mifepristone on meningioma cells was first demonstrated in 1986.44 In 1994, in vitro and in vivo effects of the progesterone antagonists, mifepristone and onapristone, were analyzed on meningioma tissues isolated from 13 patients.45 It was shown that both progesterone antagonists acted cytostatically and cytotoxically on meningioma cells in vitro to In all, 4 of 4 PR-positive meningiomas were inhibited by 108 M mifepristone and onapristone at the ninth day, respectively. On the contrary, 3 of 9 (33.3%) of PR-negative meningiomas showed complete resistance to the same doses of mifepristone and onapristone at the same period of short incubation. Hence, it is likely that PR may only partially contribute to responses against mifepristone.45 In meningiomas growing inside the renal capsules of nude mice, antiprogestins also suppressed growth, but contrary to in vitro findings, no necrotic or apoptotic cytotoxicities accompanied the growth suppression seen in vivo.45 The first clinical application of mifepristone in meningiomas was performed in 1991 and 200 mg/d mifepristone was given to 14 patients with unresectable tumors.46 The treatment durations changed between 2 and 31þ months (including treatments for longer than 12 months in 6 patients). In 5 cases (36%), objective responses were confirmed, and in 3 additional patients (21%) subjective responses were witnessed with improved extraocular muscle function and resolution of headaches.46 Side effects were mild, including fatigue in 11, hot flushes in 5, gynecomastia in 3, partial alopecia in 2, and lastly, cessation of menstruation in 2 cases, respectively. In 2 succeeding publications, Lamberts et al47,48 reported treatment responses and side effects in 12 recurrent or inoperable patients with meningioma, who received mifepristone 200 mg/d for 1 year. In patients in whom side effects occurred most prominently, hypothalamo- pituitary-adrenal axis stimulation occurred at highest levels as revealed by increased blood and urinary cortisol levels.47 Disease stabilization was seen in 6 of 10 patients who had progressive tumor growth before treatment, and in 3 of them, tumor shrinkage was evidenced radio- logically.48 In 2005, side effects of very long-term mifepristone therapy were reported in 25 patients aged between 22 and 80 years.49 Following diagnosis of unresectable meningioma, 5 patients received mifepristone between 4 and 10 months, 8 between 1 and 4 years, 5 between 4 and 9 years, and 6 between 10 and 12 years, respectively.49 In 1 premenopausal and in 1 postmenopausal patient, endometrial hyperplasia developed; whereas in 2 other patients, endometrial thickening occurred without hyperplasia.49 Hematological abnormalities or deterioration in liver and renal functions did not occur in any patients.49 Most recently in 2014, results of mifepristone therapy were reported in 3 women with multiple meningioma.50 The authors referred to previous studies that showed that diffuse meningiomatosis or multiple meningiomas expressed higher percentages of PR51 and they based their treatment on this fact. They observed that clinical responses occurred in all 3 patients and radiological responses in 2 patients. Furthermore, meningiomas had a stable course until the time of publication in all patients who received the treatment between 5 and 9 years.50 These findings are of essential importance as multiple sporadic meningiomas (but not familial multiple meningiomas) associate with NF-2/merlin mutations,52 and NF-2/merlin mutant meningiomas are genotypi- cally and phenotypically aggressive.53 These facts may be considered as paradoxical at first glance, as PR-positivity is generally seen in low-grade meningiomas. Nonetheless, this seeming paradox is explainable. The NF-2/merlin mutations enhance genomic instability by downregulating the genomic gatekeeper and proapoptotic gene p53.54 During the early stages of meningioma tumorigenesis, NF-2/merlin mutant and PR-expressing cells may be the same with relatively lesser aggressive morphology. However, more aggressive cell clones may manifest 236 İ. Elmaci et al. / Curr Probl Cancer 40 (2016) 229–243 when NF-2/merlin mutations cause survival and selection of cells resistant to chemotherapy and radiotherapy owing to deficient p53 pathway. Hence, mifepristone and HU may synergize in eradicating cell clones responsible for tumor recurrence. In a recently published clinical study, mifepristone failed to show activity in unresectable meningiomas.55 Several facts may account for these results. The main inclusion criteria in this study is that the meningiomas were surgically unresectable, which may mostly associate with anatomical difficulties rather than tumor biology itself.55 Hence, high-grade meningiomas with prominent angiogenesis may still be blocked by high doses of mifepristone, whereas those having higher proliferation indices may be eradicated with HU. Furthermore, as would be outlined below, mifepristone may allow in applying curatively high doses of HU via its myeloprotective efficacies.

Mifepristone's PR agonist activity

We have outlined earlier that the presence of PR is associated with better differentiation and lower grades of meningioma. Thus, it may also be possible that mifepristone induces a more- differentiated pattern of meningioma cells via supersaturation of PR. Indeed, mifepristone was also shown to act as a PR agonist.56 Mifepristone exerts similar activities to another progesterone agonist, medroxyprogesterone acetate (MPA). Low doses of MPA increases risk of breast cancer when employed in hormone replacement therapy of postmenopausal women.57 On the contrary, patients with breast cancer who used hormone replacement therapy had significantly lower- stage tumors and higher survival than nonusers.58 Furthermore, MPA at very high dosages act in an anticancer manner in advanced breast cancer.59,60 Therefore, employing high doses of mifepristone for long periods may trigger cellular signals similar to those activated by MPA. As would be discussed, MPA acts as a myeloprotective agent and enhances the therapeutic window for cancer chemotherapy. Despite mifepristone not having been tested against chemotherapy- induced marrow suppression as yet, several studies have concordantly demonstrated its myeloprotective activity in other contexts. Hence, we propose that the myeloprotective effects of mifepristone may enhance tolerability and therapeutic window of HU in high-grade meningiomas. Here, we would first delineate PR agonist activity of mifepristone and then the MPA enhancement of chemotherapy tolerability. Mifepristone induces binding to a progestin-responsive element of homodimers and heterodimers of the human PR (hPR) isoforms A and B, present in T47-D breast cancer cells or in HeLa cells expressing recombinant proteins.56 The resulting complexes were indistin- guishable from those induced with the progesterone analogue promegestone with respect to specificity, affinity, and stability.56 The analyses of PRs revealed the presence of 2 transcription activation functions (TAFs), located in the N-terminal region of PR-A and PR-B (TAF-1) and the hormone-binding domain (TAF-2). Both TAFs are activated in HeLa cells in the presence of promegestone.56 In the presence of mifepristone, TAF-2 was inactive, whereas TAF-1 within the PR-B-mifepristone complex activated transcription from a reporter gene containing a single progestin-responsive element. In contrast to PR-B, PR-A was not able to activate transcription in the presence of mifepristone.56 In vivo competition between PR-mifepristone and either PR- promegestone or the glucocorticoid receptor-dexamethasone complex further supported that PR-mifepristone bound in vivo to its cognate responsive element.56 Considering the instance that cessation of progestin use caused a striking regression of multiple meningiomas overexpressing PR-B37 and that the mifepristone activates transcription via PR-B,56 we hypothesize that mifepristone at high doses may also act like a progesterone analogue to stimulate differentiation in meningiomas.

Antiangiogenic activity of mifepristone

The first studies indicating antiangiogenic potential of mifepristone were performed in primates.61 In endometrial glandular epithelium, expression of VEGF was higher in progesterone-exposed İ. Elmaci et al. / Curr Probl Cancer 40 (2016) 229–243 237 endometria compared with mifepristone-exposed endometria or endometria from anovulatory cycles. Furthermore, mifepristone abolished VEGF staining in glandular epithelium almost completely.61 In another primate study, mifepristone reduced basic fibroblast growth factor levels in endometrium.62 As VEGF and basic fibroblast growth factor are major growth factors in angiogenesis, these findings indicate antiangiogenic potential of mifepristone. Then, it was shown that progestins induce VEGF release of T47-D human breast cancer cells and that mifepristone could block this increase.63,64 In gastric cancer models, mifepristone blocked VEGF release in vitro and VEGF expression and MVD in nude mice tumor xenografts in vivo, indicating that angiogenic actions of mifepristone are not limited to neutralization of progesterone effects.65 Indeed, mifepristone-exposed ovarian follicles display reduced production of VEGF in theca cells, a reduced vascular area, and a lower rate of endothelial cell proliferation.66 It was also revealed that mifepristone employed for early pregnancy termination, induces cell death in capillary endothelial cells of endometrium.67 Altogether, these findings reveal antiangiogenic effects of mifepristone both in benign and malignant tissues.

PR agonist MPA is myeloprotective against cancer chemotherapy: Role of proliferation inhibition in myeloid series

Addition of high-dose MPA to chlorambucil chemotherapy in 28 patients significantly reduced incidence, entity, and duration of the leukopenia as compared with 29 patients who received only chlorambucil.68 In 18 women with advanced breast cancer treated with MPA of 1000 mg p.o. daily, marrow granulocyte-macrophage colony-forming units (GM-CFU) declined progressively; despite that, blood granulocytes showed a modest increase.69 It was concluded that temporary blockage of cell proliferation with MPA may have rendered marrow cells resistant to chemotherapy, as classical antineoplastics mostly target proliferating cells.69 A multicentric controlled trial analyzed effects of high-dose MPA on hematological toxicity of chemotherapy.70 Combination chemotherapy (CT) conventionally employed for the various types of tumor (breast, colorectal, lung, and other solid tumors) involved, was associated with MPA (117 patients) or placebo (110 patients). MPA was given orally as a dose of 500 mg b.i.d. for 6 months. The incidence of leukopenia was significantly lower in the groups receiving MPA in patients with breast and colorectal cancer (P o 0.02), whereas tumors of the lung and other solid forms showed no such difference.70 Objective responses (CR þ partial remission) were observed in 50% of patients with breast cancer treated with CT þ MPA, and 28% of them were given CT þ placebo (P o 0.02). Contrary to expectations, posttreatment findings indicated increases in antithrombin III and plasminogen, which suggest that MPA does not increase the risk of thrombosis, and might even, to some extent, impede cancer thrombophilia.70 Results of high-dose MPA addition to cyclophosphamide-Adriamycin-cisplatin (CAP) chemotherapy in patients with gynecological cancers have also been reported.71 It was revealed that (1) leukocyte count reached minimum at the second week of CAP with no significant difference of MPA addition.71 However, at the third week of chemotherapy, the count improved to 84% of the pre-CAP level in patients receiving MPA and to 68% in those not receiving MPA (P o 0.01).71 At the fourth week, the leukocyte count returned to the pre-CAP level only in patients receiving MPA. (2) In patients receiving MPA, platelet count improved more rapidly within 3 weeks (P o 0.01), and was significantly higher at the fourth week (P o 0.05).71 (3) The period needed for 1 course of CAP were shorter in patients receiving adjuvant MPA (P o 0.05).71 These results show that MPA accelerates recovery from marrow depression, which may prove to augment chemotherapy efficacy via acceleration of phases or application of higher dosages. In another study, 21 patients with head and neck cancer were randomized into 2 arms—11 patients received multiagent chemotherapy including cisplatin, methotrexate, bleomycin, and vincris- tine; and 10 patients were treated with the same schedule plus 1000 mg/d of MPA.72 MPA was administered 14 days before chemotherapy and continued daily up to the 90th day. Bone marrow was harvested on 0, 14, and 90 days after chemotherapy. Diverse GM-CFU behavior in the 2 arms supported the hypothesis that the MPA myeloprotectivity effect is due to a mitotic rest in the marrow stem cells.72 238 İ. Elmaci et al. / Curr Probl Cancer 40 (2016) 229–243

Effects of MPA on antitumor efficacy and side effects of 5-fluorouracil (5-FU) were investigated in ddY mice.73 5-FU acts as a thymidylate synthase inhibitor and blocks synthesis of thymidine required for DNA synthesis. Interestingly, MPA reduced lethal toxicity, body weight loss, and the bone marrow toxicity of 5-FU in aged female ddY mice, which was not observed in young male ddY mice.73 Moreover, MPA did not hinder antitumor activity of 5-FU against MPA- insensitive mouse breast carcinoma FM3A; indeed it augmented it against MPA-sensitive human breast carcinoma MCF-7.73 As 5-FU acts primarily via inhibiting DNA synthesis, these findings are of particular importance for our proposal to combine mifepristone with HU, which also functions as a DNA synthesis inhibitor. In human marrow cells too, MPA acted more protective against S-phase–specific drugs such as 5-FU.74 The percentage of GM-CFU in S-phase was significantly reduced from 67% 7 2.5% (control cells) to 38% 7 5.5% (24-hour MPA- preincubated cells).74 When human bone marrow cells were incubated for 3 days with MPA (100 ng/mL) and then exposed to adriamycin, LD50 of adriamycin was significantly increased in MPA-preincubated cells (896 ng/mL) vs control cells (162 ng/mL).74 Particularly noteworthy was the finding that MPA did not protect human leukemic cell lines from adriamycin cytotoxicity, suggesting prominent potential of MPA to uncouple chemotherapy risks and benefits.74 Lastly, endogenous cytokine release and marrow cellularity of 15 patients with cancer were investigated to elucidate effects of high-dose MPA on chemotherapy in neutropenia.75 MPA (1 g/d p.o.) was started after the first cycle of chemotherapy in patients with neutropenia and was stopped 1 week after the second cycle of chemotherapy. Marrow samples were obtained for granulocyte-macrophage colony-stimulating factor assay 1 week after chemotherapy cycles and were compared before and after MPA treatment.75 Of 15 patients, 12 patients had a significant decrease in granulocyte-macrophage colony-stimulating factor secretion after MPA treatment.75 These results revealed that the MPA reduction of marrow progenitor cell proliferation is mediated by suppression of marrow growth factors.

Mifepristone blocks glucocorticoid-induced apoptosis of B-cell progenitors and mitogen- induced proliferation of neutrophil progenitors

Mifepristone could block glucocorticoid-induced apoptosis of (CD10þCD19þ) B-lymphocyte progenitors76,77 and senescence of mesenchymal stromal cells.77–79 Mifepristone could also block lactoferrin-induced leukocytosis and increase of the myelocytic lineage cells.80 Lactoferrin increases blood leukocytes by 49% (P o 0.05), which accompanies significant increases in the blood level of neutrophils (2-fold) and neutrophil precursors or bands (10-fold) in mice.80 These results are of considerable importance, as mifepristone reduction of actively proliferating myeloid precursors (and also mature neutrophils are likely subsequent to progenitor cell blockage) resembles the effects exerted by MPA. Hence, mifepristone at high dosage may block active proliferation of myeloid precursor cells similar to MPA, which could reduce the severity and extent of neutropenic intervals during chemotherapy. We have mentioned earlier that MPA did not hinder antitumor efficacy of S-phase–specific chemotherapeutics despite protecting the marrow cells. This sensitive modification would be because of the differential cell proliferation kinetics of marrow and solid tumor cells. Here, marrow progenitor cells with a higher population turnover may be more sensitive to chemotherapy, and slowering this process may significantly reduce myelotoxicity. The main similarity between mifepristone and MPA mentioned here is their ability to reduce marrow cell proliferation rates, despite having mainly opposite actions on sex steroid–sensitive tumors.

Isoform-speciﬁc roles of PR in cellular growth and agonist-dependent downregulation of PRs: Synergism of mifepristone and HU may occur both in the absence and presence of the PR

PR is present on normal meningeal cap cells, whereas meningioma cell growth is stimulated by exogenous progesterone analogues or with pregnancy. Absence of or reduction in PRs with İ. Elmaci et al. / Curr Probl Cancer 40 (2016) 229–243 239 increasing grade of meningiomas is also indicative of progesterone having a dual function on meningioma growth—a proliferating function and a differentiation function. Supraphysiological dosages of progesterone analogues may shift the balance of proliferation and differentiation toward higher differentiation via changing the expression ratio of different PR isoforms, PR-A and PR-B. Moreover, the different PR isoforms may exert opposite functions in different tissues. For instance, in placental tissues, PR-A blocks NF-κB activity and COX-2 expressions, which associate with antiapoptotic, proliferative, and proinflammatory actions, all related with protumorigenic effects.81 On the contrary in leiomyoma cells, progesterone decreases the proliferative IGF-I protein via activating PR-B.82 Similarly, preventive and inhibitory efficacy of progesterone against endometrial cancer occurs via PR-B.83 It is known that many cellular receptors are downregulated in the sustained presence of their ligands, which is also relevant for PR.84 However, even this would be beneficial with regard to blocking meningioma growth, as it was demonstrated that efficient treatment of endometrial hyperplasia by the levonorgestrel (a progesterone analogue)-impregnated intrauterine device was associated with downregulation of both PR-A and PR-B.85 Considering the long-term continuing exposure of hyperplastic endometrium to progestin in this process, it may be possible that long-term antitumor efficacy of progestins occurs via downregulating all of their receptors. In that case, PR-negative meningioma clones (which may be less differentiated or exerting stem cell features or both) emerge after long periods of treatment. Even then, additional treatment with HU would be of value, as DNA synthesis inhibitory agents are more efficient in rapidly cycling hormone receptor–negative tumors. Furthermore, HU may delay development of hormone receptor–negative clones, as a prominent elevation in nuclear glucocorticoid receptor binding of [3H]dexamethasone was seen in peripheral leukocytes of patients with leukemia following treatment with HU plus prednisone, but not after treatment with prednisone alone.86 HU potentiation of glucocorticoid activity is also observed in hypereosinopilic syndrome that can transform into eosinophilic leukemia. In steroid-unresponsive hypereosinopilic syndrome, addition of HU to steroid treatment achieved CR.87,88 In experimental leukemia models too, HU potentiated both the antiproliferative and differentiating functions of retinoic acid and vitamin D, which both bind to steroid-thyroid hormone receptor superfamily receptors similar to progestins.89 In breast cancer cells, antiestrogens hinder transcription of ERα target genes specifically in S-phase, and their influence on cell proliferation is higher in HU-arrested cells.90

Ribonucleotide reductase subunit 2 and RRM2 and calcium channels as shared targets and modiﬁers of mifepristone and HU

Lastly, 2 other crucially important features exist indicating the potential of HU and mifepristone synergism in the treatment of WHO grade II and III meningioma. Very recent microarray analysis revealed that during embryogenesis, ribonucleotide reductase type-II subunit and RRM2 expression was signiﬁcantly higher at implantation sites compared with interimplantation sites in mouse uterus.91 Although both RRM1 and RRM2 expression are signiﬁcantly high in mouse uterine stromal cells undergoing decidualization, only RRM2 is induced by progesterone, a key regulator of decidualization.91 Further analyses demonstrated that the stimulation of progesterone on RRM2 expression in stromal cells is mediated by the AKT/c-MYC pathway.91 Hence, antagonism (or augmentation of the differentiating activity) of PR via mifepristone or high and sustained dose progestin-induced PR downregulation would potently block the RR pathway, via both reduction of expression and enzyme activity. Relatively recently, it was shown that calcium channel–blocking agents (diltiazem and verapamil) could augment antitumor activity of both mifepristone and HU on meningioma cells in vitro and in vivo.92 The addition of diltiazem or verapamil to HU or RU486 potentiated meningioma (IOMM- Lee) growth inhibition by 20%-60% in vitro via apoptosis and G1 cell cycle arrest.92 In vivo, tumors treated with this combination of drugs were smaller, and immunohistochemical analysis of the IOMM-Lee tumors showed an approximate 75% decrease in microvascular density.92 240 İ. Elmaci et al. / Curr Probl Cancer 40 (2016) 229–243

A recent strong proof pertinent to the involvement of progesterone pathway in multiple meningiomas and meningiomatosis

As cyproterone acetate (CA), a progesterone agonist, was observed to exert a stronger influence on meningioma growth, all patients for a suspicion of meningioma were questioned specifically about exogenous sex hormone intake and more specifically about CA intake for a recent period of 5 years.93 In total, 12 patients who were taking CA were defined, and very noteworthy, 10 of these with a more prolonged usage had multiple meningiomas.93 Discon- tinuation of CA caused tumor shrinkage in 11 patients and stopped tumor growth in 1 with no regrowth during a mean follow-up period of 12 months (range: 5-35 months).93

Conclusions

Meningiomas constitute approximately one-third of all brain tumors, and approximately 20% meningiomas are high-grade neoplasms causing signiﬁcant morbidity and mortality. Despite surgical treatment yielding signiﬁcantly good clinical outcomes in WHO grade I meningiomas, high-grade meningiomas frequently recur and invade into critical anatomical locations hindering complete tumor removal. Hitherto, a golden standard of pharmacological management has not been described for these tumors. Both HU and mifepristone are shown to slow meningioma growth to some extent and the proposed synergistic pathways for mifepristone and HU are summarized in the Figure. Owing to earlier outlined facts, we believe that combining mifepristone with high doses of HU may lead to improved tumor control, lesser systemic toxicity, and higher survival in patients suffering from these grave malignancies.

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