NGF/TRKA Promotes ADAM17-Dependent Cleavage of P75 in Ovarian Cells: Elucidating a Pro-Tumoral Mechanism.

Nerve growth factor (NGF) and its high-affinity receptor TRKA are overexpressed in epithelial ovarian cancer (EOC) displaying a crucial role in the disease progression. Otherwise, NGF interacts with its low-affinity receptor P75, activating pro-apoptotic pathways. In neurons, P75 could be cleaved by metalloproteinases (α and γ-secretases), leading to a decrease in P75 signaling. Therefore, this study aimed to evaluate whether the shedding of P75 occurs in EOC cells and whether NGF/TRKA could promote the cleavage of the P75 receptor. The immunodetection of the α-secretase, ADAM17, TRKA, P75, and P75 fragments was assessed by immunohisto/cytochemistry and Western blot in biopsies and ovarian cell lines. The TRKA and secretases' inhibition was performed using specific inhibitors. The results show that P75 immunodetection decreased during EOC progression and was negatively correlated with the presence of TRKA in EOC biopsies. NGF/TRKA increases ADAM17 levels and the fragments of P75 in ovarian cells. This effect is abolished when cells are previously treated with ADAM17, γ-secretase, and TRKA inhibitors. These results indicate that NGF/TRKA promotes the shedding of P75, involving the activation of secretases such as ADAM17. Since ADAM17 has been proposed as a screening marker for early detection of EOC, our results contribute to understanding better the role of ADAM17 and NGF/TRKA in EOC pathogenesis, which includes the NGF/TRKA-mediated cleavage of P75.

The 5' untranslated region of the anti-apoptotic protein Survivin contains an inhibitory upstream AUG codon.

Survivin (BIRC5) is an anti-apoptotic protein that is important in cancer. Mechanisms responsible for controlling Survivin levels in cells include transcriptional regulation and modulation of protein stability via post-translational modifications; however to date, translational control has been poorly studied. Here, we focused particularly on the primary control elements present in the Survivin 5' untranslated region (5'UTR). Bioinformatic analysis of ribosome occupancy on the Survivin 5'UTR revealed the presence of elongating ribosomes upstream of the canonical initiator AUG, suggesting an alternative upstream initiator AUG (uAUG) might exist. This uAUG was found out-of-frame at position -71 and appeared as a conserved element in mammals. RACE analysis revealed different transcriptional start sites for BIRC5, which indicated that translational control by this uAUG is restricted to longer 5'UTR variants. We studied the activity of the uAUG in different cell types by cloning the Survivin 5'UTR DNA sequence (wild-type and mutated variants) upstream of renilla luciferase (RLuc) into a pcDNA3 plasmid. Changes in RLuc activity were determined by luminescence assays and Western blotting. Results showed that when this uAUG was mutated to AUU or AGG in the cloned Survivin 5'UTR, RLuc activity was significantly increased. Similar results were obtained when uAUG was positioned inframe with the RLuc initiator AUG. Immunodetection of Renilla (35 kDa) by Western blotting revealed the presence of a second band (37 kDa approximately) in cells transfected with the Inframe reporter constructs, indicating that the uAUG was functional in our experimental conditions. In conclusion, our experimental data demonstrate the presence of an alternative and inhibitory initiator uAUG in the Survivin 5' UTR. This inhibitory uAUG may help understanding how Survivin expression is downregulated under physiological or pathological conditions.

NGF/TRKA Decrease miR-145-5p Levels in Epithelial Ovarian Cancer Cells.

Nerve Growth Factor (NGF) and its high-affinity receptor tropomyosin receptor kinase A (TRKA) increase their expression during the progression of epithelial ovarian cancer (EOC), promoting cell proliferation and angiogenesis through several oncogenic proteins, such as c-MYC and vascular endothelial growth factor (VEGF). The expression of these proteins is controlled by microRNAs (miRs), such as miR-145, whose dysregulation has been related to cancer. The aims of this work were to evaluate in EOC cells whether NGF/TRKA decreases miR-145 levels, and the effect of miR-145 upregulation. The levels of miR-145-5p were assessed by qPCR in ovarian biopsies and ovarian cell lines (human ovarian surface epithelial cells (HOSE), A2780 and SKOV3) stimulated with NGF. Overexpression of miR-145 in ovarian cells was used to evaluate cell proliferation, migration, invasion, c-MYC and VEGF protein levels, as well as tumor formation and metastasis in vivo. In EOC samples, miR-145-5p levels were lower than in epithelial ovarian tumors. Overexpression of miR-145 decreased cell proliferation, migration and invasion of EOC cells, changes that were concomitant with the decrease in c-MYC and VEGF protein levels. We observed decreased tumor formation and suppressed metastasis behavior in mice injected with EOC cells that overexpressed miR-145. As expected, ovarian cell lines stimulated with NGF diminished miR-145-5p transcription and abundance. These results suggest that the tumoral effects of NGF/TRKA depend on the regulation of miR-145-5p levels in EOC cells, and that its upregulation could be used as a possible therapeutic strategy for EOC.

Effects of sympathectomy on ovarian follicular development and steroid secretion.

Recently, the influence of adrenergic activity over ovarian function, and thus fertility, has begun to gain importance. Previous studies have shown that adrenergic activity through norepinephrine (NE) participates in the control of follicular development and steroidal secretion from the ovary, among other functions. To examine this phenomenon, the denervation of the gonad has been widely used to observe changes in the ovary's performance. Nevertheless, the effect of the absence of adrenergic nerves in the ovary has only been studied in short times periods. In the present work, we used guanethidine (a drug that produces an irreversible sympathectomy) during the infantile period of rats, and we observed its effects in the adult rat (6 months old). Our results indicate that ovarian NE content is recovered at 6 months old, alongside with an increase of the adrenal content of NE and a dysfunctional celiac ganglion. Together, these results suggest that the recovery of ovarian NE does not come from a neural origin. In addition, ovarian performance was impaired because the changes in follicular development and steroidal secretion are not recovered despite the recovery of ovarian NE content. In conclusion, these results suggest that the nerve-ovarian connections, which are established during infantile development, are necessary for the accurate response of the ovary to sympathetic stimulation.

Mechanisms of Regulation of the Expression of miRNAs and lncRNAs by Metformin in Ovarian Cancer.

Ovarian cancer (OC) is one of the most lethal gynecological malignancies. The use of biological compounds such as non-coding RNAs (ncRNAs) is being considered as a therapeutic option to improve or complement current treatments since the deregulation of ncRNAs has been implicated in the pathogenesis and progression of OC. Old drugs with antitumoral properties have also been studied in the context of cancer, although their antitumor mechanisms are not fully clear. For instance, the antidiabetic drug metformin has shown pleiotropic effects in several in vitro models of cancer, including OC. Interestingly, metformin has been reported to regulate ncRNAs, which could explain its diverse effects on tumor cells. In this review, we discuss the mechanism of epigenetic regulation described for metformin, with a focus on the evidence of metformin-dependent microRNA (miRNAs) and long non-coding RNA (lncRNAs) regulation in OC.

Conventional and new proposals of GnRH therapy for ovarian, breast, and prostatic cancers.

For many years, luteinizing hormone-releasing hormone or gonadotropin-releasing hormone (GnRH) analogs have been used to treat androgen or estrogen-dependent tumors. However, emerging evidence shows that the GnRH receptor (GnRH-R) is overexpressed in several cancer cells, including ovarian, endometrial, and prostate cancer cells, suggesting that GnRH analogs could exert direct antitumoral actions in tumoral tissues that express GnRH-R. Another recent approach based on this knowledge was the use of GnRH peptides for developing specific targeted therapies, improving the delivery and accumulation of drugs in tumoral cells, and decreasing most side effects of current treatments. In this review, we discuss the conventional uses of GnRH analogs, together with the recent advances in GnRH-based drug delivery for ovarian, breast, and prostatic cancer cells.

Role of Mitochondria in Interplay between NGF/TRKA, miR-145 and Possible Therapeutic Strategies for Epithelial Ovarian Cancer.

Ovarian cancer is the most lethal gynecological neoplasm, and epithelial ovarian cancer (EOC) accounts for 90% of ovarian malignancies. The 5-year survival is less than 45%, and, unlike other types of cancer, the proportion of women who die from this disease has not improved in recent decades. Nerve growth factor (NGF) and tropomyosin kinase A (TRKA), its high-affinity receptor, play a crucial role in pathogenesis through cell proliferation, angiogenesis, invasion, and migration. NGF/TRKA increase their expression during the progression of EOC by upregulation of oncogenic proteins as vascular endothelial growth factor (VEGF) and c-Myc. Otherwise, the expression of most oncoproteins is regulated by microRNAs (miRs). Our laboratory group reported that the tumoral effect of NGF/TRKA depends on the regulation of miR-145 levels in EOC. Currently, mitochondria have been proposed as new therapeutic targets to activate the apoptotic pathway in the cancer cell. The mitochondria are involved in a myriad of functions as energy production, redox control, homeostasis of Ca+2, and cell death. We demonstrated that NGF stimulation produces an augment in the Bcl-2/BAX ratio, which supports the anti-apoptotic effects of NGF in EOC cells. The review aimed to discuss the role of mitochondria in the interplay between NGF/TRKA and miR-145 and possible therapeutic strategies that may decrease mortality due to EOC.

Current Treatments and New Possible Complementary Therapies for Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) is one of the deadliest gynaecological malignancies. The late diagnosis is frequent due to the absence of specific symptomatology and the molecular complexity of the disease, which includes a high angiogenesis potential. The first-line treatment is based on optimal debulking surgery following chemotherapy with platinum/gemcitabine and taxane compounds. During the last years, anti-angiogenic therapy and poly adenosine diphosphate-ribose polymerases (PARP)-inhibitors were introduced in therapeutic schemes. Several studies have shown that these drugs increase the progression-free survival and overall survival of patients with ovarian cancer, but the identification of patients who have the greatest benefits is still under investigation. In the present review, we discuss about the molecular characteristics of the disease, the recent evidence of approved treatments and the new possible complementary approaches, focusing on drug repurposing, non-coding RNAs, and nanomedicine as a new method for drug delivery.

Follicle-stimulating hormone promotes nerve growth factor and vascular endothelial growth factor expression in epithelial ovarian cells.

Ovarian cancer is the first cause of death for gynecological malignances in developed countries and around 80% correspond to Epithelial Ovarian Cancer (EOC). Overexpression of Nerve Growth Factor (NGF) and its high affinity receptor TRKA are involved in EOC progression, modulating several oncogenic processes such as angiogenesis by the increase of Vascular Endothelial Growth Factor (VEGF). FSH receptors (FSH-R) are present in EOC, but their changes and contribution during EOC progression are still not thoroughly known. The aims of this study were to evaluate the abundance of FSH receptors during EOC differentiation and to determine whether FSH modulates oncoproteins such as NGF and VEGF in ovarian cells. FSH-R expression in EOC tissues and cell lines (A2780, poorly differentiated EOC cells and HOSE, non-tumoral ovarian surface epithelial cells) were measured by RT-PCR and laser capture of epithelial cells from EOC samples by qPCR. FSH-R protein levels were evaluated by immunohisto/cytochemistry. Additionally, ovarian explants and ovarian cell lines were stimulated with FSH and/or FSH-R inhibitor to assess NGF and VEGF mRNA and protein levels. The results showed that FSH-R levels decreased during EOC progression, nevertheless these receptors are still present in poorly differentiated EOC. FSH increased NGF expression in ovarian cells, which was prevented using a FSH-R inhibitor. Similarly, in ovarian cancer explants, FSH increased NGF and VEGF mRNA, as well as NGF protein levels. These results suggest that FSH would display a key role not only in initial stages of EOC, but also in late stages of this disease, by modulation of NGF and VEGF levels in EOC cells.

Metformin Reduces NGF-Induced Tumour Promoter Effects in Epithelial Ovarian Cancer Cells.

Epithelial ovarian cancer (EOC) is a lethal gynaecological neoplasm characterized by rapid growth and angiogenesis. Nerve growth factor (NGF) and its high affinity receptor tropomyosin receptor kinase A (TRKA) contribute to EOC progression by increasing the expression of c-MYC, survivin and vascular endothelial growth factor (VEGF) along with a decrease in microRNAs (miR) 23b and 145. We previously reported that metformin prevents NGF-induced proliferation and angiogenic potential of EOC cells. In this study, we sought to obtain a better understanding of the mechanism(s) by which metformin blocks these NGF-induced effects in EOC cells. Human ovarian surface epithelial (HOSE) and EOC (A2780/SKOV3) cells were stimulated with NGF and/or metformin to assess the expression of c-MYC, ß-catenin, survivin and VEGF and the abundance of the tumor suppressor miRs 23b and 145. Metformin decreased the NGF-induced transcriptional activity of MYC and ß-catenin/T-cell factor/lymphoid enhancer-binding factor (TCF-Lef), as well as the expression of c-MYC, survivin and VEGF in EOC cells, while it increased miR-23b and miR-145 levels. The preliminary analysis of ovarian biopsies from women users or non-users of metformin was consistent with these in vitro results. Our observations shed light on the mechanisms by which metformin may suppress tumour growth in EOC and suggest that metformin should be considered as a possible complementary therapy in EOC treatment.

Increases in norepinephrine release and ovarian cyst formation during ageing in the rat.

BACKGROUND: Depletion of ovarian follicles is associated with the end of reproductive function in ageing females. Recently, it has been described that this process parallels increases in the concentration of norepinephrine (NE) in the rat ovary. In sexually mature rats, experimentally-induced increases in the sympathetic tone of the ovary is causally related to ovarian cyst formation and deranged follicular development. Thus, there is a possibility that increased ovarian NE concentrations represent changes in the activity of sympathetic nerves, which consequently participate in the process of ovarian cyst formation observed during ageing in the human and experimental animal models. METHODS: Sprague-Dawley rats between 6 and 14 months old were used to analyse the capacity of the ovary to release 3H-NE recently incorporated under transmural depolarisation in relation to changes in the ovarian follicular population. Morphometric analysis of ovarian follicles and real time PCR for Bcl2 and Bax mRNA were used to assess follicular atresia. RESULTS: From 8 months old, the induced release of recently incorporated 3H-norepinephrine (3H-NE) from the ovary and ovarian NE concentrations increased, reaching their peak values at 12 months old and remained elevated up to 14 months old. Increases in sympathetic nerve activity paralleled changes in the follicular population, as well as disappearance of the corpus luteum. In contrast, luteinised follicles, precystic follicles, and cystic follicles increased. During this period, the relationship between Bax and Bcl2 mRNAs (the proapoptotic/antiapoptotic signals) increased, suggesting atresia as the principal mechanism contributing to the decreased follicular population. When NE tone was increased, the mRNA ratio favoured Bcl2 to Bax and antiapoptotic signals dominated this period of development. Thus, these changing ratios could be responsible for the increase in luteinised follicles, as well as precystic and cystic follicles. CONCLUSION: These data suggest that the ageing process in the ovary of the Sprague-Dawley rat is accompanied by an increased sympathetic tone of the ovary. Consequently, this sympathetic change could be related to a neuroendocrine-driven formation of a polycystic condition similar to that observed in the sympathetic-activated adult ovary.

Angiogenesis in Gynecological Cancers: Role of Neurotrophins.

Angiogenesis, or generation of new blood vessels from other pre-existing, is a key process to maintain the supply of nutrients and oxygen in tissues. Unfortunately, this process is exacerbated in pathologies such as retinopathies and cancers with high angiogenesis as ovarian cancer. Angiogenesis is regulated by multiple systems including growth factors and neurotrophins. One of the most studied angiogenic growth factors is the vascular endothelial growth factor (VEGF), which is overexpressed in several cancers. It has been recently described that neurotrophins could regulate angiogenesis through direct and indirect mechanisms. Neurotrophins are a family of proteins that include nerve growth factor (NGF), brain-derived growth factor (BDNF), and neurotrophins 3 and 4/5 (NT 3, NT 4/5). These molecules and their high affinity receptors (TRKs) regulate the development, maintenance, and plasticity of the nervous system. Furthermore, it was recently described that they display essential functions in non-neuronal tissues, such as reproductive organs among others. Studies have shown that several types of cancer overexpress neurotrophins such as NGF and BDNF, which might contribute to tumor progression and angiogenesis. Besides, in recent years the FDA has approved the use of pharmacologic inhibitors of pan-TRK receptors in patients with TRKs fusion-positive cancers. In this review, we discuss the mechanisms by which neurotrophins stimulate tumor progression and angiogenesis, with emphasis on gynecological cancers.

NGF-Enhanced Vasculogenic Properties of Epithelial Ovarian Cancer Cells Is Reduced by Inhibition of the COX-2/PGE2 Signaling Axis.

Epithelial ovarian cancer (EOC) is a lethal gynecological neoplasia characterized by extensive angiogenesis and overexpression of nerve growth factor (NGF). Here, we investigated the mechanism by which NGF increases vascular endothelial growth factor (VEGF) expression and the vasculogenic potential of EOC cells, as well as the contribution of the cyclooxygenase 2/prostaglandin E2 (COX-2/PGE2) signaling axis to these events. EOC biopsies and ovarian cell lines were used to determine COX-2 and PGE2 levels, as well as those of the potentially pro-angiogenic proteins c-MYC (a member of the Myc transcription factors family), survivin, and ß-catenin. We observed that COX-2 and survivin protein levels increased during EOC progression. In the EOC cell lines, NGF increased the COX-2 and PGE2 levels. In addition, NGF increased survivin, c-MYC, and VEGF protein levels, as well as the transcriptional activity of c-MYC and ß-catenin/T-cell factor/lymphoid enhancer-binding factor (TCF-Lef) in a Tropomyosin receptor kinase A (TRKA)-dependent manner. Also, COX-2 inhibition prevented the NGF-induced increases in these proteins and reduced the angiogenic score of endothelial cells stimulated with conditioned media from EOC cells. In summary, we show here that the pro-angiogenic effect of NGF in EOC depends on the COX-2/PGE2 signaling axis. Thus, inhibition COX-2/PGE2 signaling will likely be beneficial in the treatment of EOC.

Metformin prevents nerve growth factor-dependent proliferative and proangiogenic effects in epithelial ovarian cancer cells and endothelial cells.

BACKGROUND: Epithelial ovarian cancer (EOC) is characterized by exacerbated angiogenesis regulated by proangiogenic and growth factors. Nerve growth factor (NGF) is overexpressed in EOC where it promotes proliferation as well as survival and is considered a proangiogenic factor. Metformin, a drug commonly used in the treatment of diabetes, is attributed to antineoplastic effects, but the underlying mechanisms remain unknown. Given that current therapies yield modest results in EOC patients, the aim of this study was to determine the effects of metformin on NGF-enhanced proliferation of EOC cells and the angiogenic potential of endothelial cells. METHODS: A2780 (EOC), HOSE (human ovarian surface epithelial) and EA.hy926 (endothelial) cells were treated with NGF and metformin. Cell viability, cell proliferation and cell cycle were evaluated in all three cell lines, and the angiogenic potential in endothelial EA.hy926 cells. RESULTS: NGF enhanced cell proliferation in A2780, HOSE and EA.hy926 cells (p < 0.05), while metformin treatment decreased cell proliferation in A2780 and EA.hy926 cells (p < 0.05). Moreover, the NGF-enhanced angiogenic score in EA.hy926 cells was prevented by metformin (p < 0.05). CONCLUSIONS: Given that NGF plays a significant role in EOC progression, our current findings suggest that metformin holds considerable promise as an adjuvant treatment in ovarian cancer.