Estrogen/Neuroglobin pathway as pharmacological target for hormone-related cancer

Abstract

In 2000, the third heme-globin, called Neuroglobin (NGB), has been discovered in nervous system. NGB comes up as a hexa-coordinated globin, but its coordination is reversible and controlled by the oxidative state of the cell as well as by phosphorylation. For this reason, NGB has been postulated to act as a redox sensor in the nervous system. Experimental works assess that NGB is a neuroprotective molecule which overexpression could protect nervous system against several insults. This data prompted several laboratories to find compounds able to increase NGB levels. Recent results showed that 17β-estradiol (E2) strongly induces NGB up-regulation by 300% in human neuroblastoma cell line. 17β-estradiol (E2) is the most concentrated and efficient female sex steroid hormone. In premenopausal women, it is produced in the ovary and its levels are significantly high during the reproductive age. E2 controls many aspects of human physiology, including development, reproduction, homeostasis and brain functions through the activity of its receptors, ERα and ERβ. Given this widespread role of E2 in human physiology, it is not surprising that it is also implicated in the development or progression of numerous diseases, which include various types of cancers (breast, ovarian, prostate and endometrial). The main factors that influence E2 effects are the state and the balance of its receptors, which levels are modified in cancer cells. ERα appears to promote the proliferation of breast, gynaecologic cancers and endocrine gland cancer cells. In contrast, ERβ suppresses the proliferation of tumor cells and drives them to apoptosis. Conversely, to what occur in extra nervous cancer tissues, in the brain ERα- and ERβ-mediated functions converge to a common outcome, neuroprotection. Previous work performed in our laboratory indicates that E2 via ERβ genomic and rapid signals exerts an anti-apoptotic and protective function in neurons by up-regulating and re-allocating NGB into mitochondria. In addition, in hepatocarcinoma and in breast cancer cells, E2 via ERα induced the up-regulation and re-localization of NGB into mitochondrial compartment in which the globin counteracts the apoptotic cascade induced by oxidative stress without any further effect on E2-induced cell proliferation, suggesting a specific role of NGB in the apoptotic pathway. In order to better define the functional role played by NGB in E2-induced cellular effects, this thesis was aimed: (i) to dissect the signalling pathways, important for cancer cell survival, which up-stream E2-induced NGB over-expression, (ii) to evidence the function played by NGB in E2-related cancer, and (iii) to screen molecules able to interfere with E2- activated pathway via ERα, in order to interfere with NGB over-expression in breast cancer cells. In the first part of this thesis, we demonstrated that NGB levels are high in human breast tumor tissues, confirming a key role of the globin in breast cancer physiology. Moreover, we showed a strong correlation between NGB and ERα. In order to obtain a more precise correlation between NGB level and ERα activation, we considered five breast cancer cell lines, characterized by a different ERα expression. NGB is expressed in all cell lines but E2 dependent NGB up-regulation is maintained only in ERα(+) breast cancer cell lines (i.e., MCF-7, T47D, ZR-75-1); while no E2 effects are reported in ERs(-) cells (i.e., SK-BR-3 and MDA-MB-231). Taken together, these data confirm a close relationship between the activation of ERα and the E2-induced NGB over-expression in breast cancer. Interesting results are obtained in ERα(-) SK-BR-3 cell line, in which high NGB amount is observed but not under E2 regulation suggesting that an active signaling pathway should exist in this cell line. Consequently, we evaluated the pathways involved. Firstly, we demonstrate that both the impairment of protein degradation and the enhancement of protein synthesis are at the root of E2-dependent rapid NGB up-regulation. Moreover, we showed that both PI3K/PDK/AKT pathway and PKC activation, an upstream activator of AKT, are required for the E2-induced increase of NGB levels in ERα(+) cell lines. In particular, E2 rapidly and persistently activates AKT phosphorylation and the pathway culminates in NGB up regulation and re-allocation into mitochondria. The high NGB level in whole cells and mitochondria paralleled with the high AKT phosphorylation status found in ERα(-) and Her2/Neu(+) SK-BR-3 cells strongly sustaining the role of AKT. Moreover, no activation of AKT and barely detectable level of NGB are reported in the triple negative MDA-MB-231 cell line. Our results indicate the crucial role of transcription factor CREB (one of the AKT downstream transcriptional factors) in the E2-induced modulation of NGB levels in both MCF-7 and T47D cells. Therefore, the rapid and persistent E2/ERα-induced AKT assure the rapid NGB accumulation into the cells, the long-term transcription of NGB gene, and the translocation of NGB into mitochondria where the globin exerts its role in promoting cell survival and avoiding the triggers of apoptotic cascade. In the second part of this thesis, we tested the effects of different stressors on the level, localization, and function of NGB in wild-type or NGB stable silenced breast cancer cells. The data indicate that hypoxia does not affect NGB protein amount in breast cancer cells, while nutrient deprivation induces an up-regulation of the globin, suggesting its possible involvement into the autophagic flux. Moreover, NGB level could be considered as a sensor of ROS being up-regulated by ROS (H2O2) and by ROS-inducing substances (Pb(IV)), which not re-allocated the globin into mitochondria. In fact, ROS and ROS-inducing compounds rapidly and transiently activate the AKT phosphorylation, unlike E2. Finally, we demonstrated that in MCF-7 cells, an active ERα is at the root of MCF-7 insensitivity to paclitaxel, a chemotherapeutic agent. In the absence of active ERα, paclitaxel significantly reduces the NGB cell content, suggesting that the NGB down-regulation could represent a mechanism by which chemotherapeutic drugs render cancer cells more prone to death. In the third part of this thesis, we screened different plant-derived polyphenols able to interfere with E2 actions. The selected compounds showed different effects on NGB and E2- induced NGB up-regulation. In particular, we selected Naringenin (Nar) and Resveratrol (Res), which are able to interfere with E2-induced NGB up-regulation; moreover, while Nar does not affect NGB level, Res decreases globin protein amount. Consequently, using ERα(+), ERs(-) and ERs(+) cell lines, we showed different effects of selected compounds on NGB and E2-induced NGB up-regulation, confirming that polyphenols act as E2 agonists and antagonists depending on the receptor content (ERα and ERβ) of specific tissues. Thus, we evaluated the Nar and Res effects on ERα and AKT activation, and demonstrated that Nar effects on decreasing E2-induced NGB levels are mediated by the inhibition of E2- induced AKT phosphorylation. Instead, Res acts through the inactivation of ERα phosphorylation and consequently, decreased AKT phosphorylation due to E2. Finally, Res and Nar, preventing E2-induced NGB up-regulation, enhance paclitaxel effect on inducing apoptosis cell death rendering cancer cell more prone to death. As a whole, these results show that E2/NGB pro-survival pathway represents a conserved activated mechanism in ERα breast cancer. Thus, we define NGB as a conserved compensatory protein induced by E2 and identify the ERα activated PI3K/AKT signaling as the main intracellular upstream pathway by which E2 affects NGB compartmentalization into mitochondria. Moreover, NGB could be considered as sensor of ROS in breast cancer. Furthermore, our data reveal the pivotal role played by NGB in the E2/ERα-induced anti apoptotic pathway that abrogates paclitaxel-induced cell death. Finally, data of this thesis indicate that polyphenols, which impairs E2-dependent induction of NGB in breast cancer cells could be useful to reduce cancer cells endurance to chemotherapeutic treatment.