New local ganirelix sustained release therapy for uterine leiomyoma. Evaluation in a preclinical organ model.

Currently, there is a limited number of treatment options available for patients with symptomatic leiomyomas, and surgical removal is by far the most frequent procedure. Previous studies found that GnRH agonists and antagonists acting through GnRH receptors led to cell death and decreased extracellular synthesis in cultured leiomyoma cells. In this study, we encapsulated the GnRH antagonist ganirelix in PLGA microspheres contained in an alginate scaffold that also supports a leiomyoma ex vivo tissue explant. Microspheres maintained ganirelix concentration stably during six days of culture, inducing significant cell death in 50-55% of tumor cells. Although no changes were observed in the expression of extracellular matrix genes, a decreased expression of the Nuclear Factor of Activated T cells 5, a transcription factor involved in osmotic stress and tumor size. Interestingly, all tumors analyzed experienced apoptosis independently of the original driver mutation. These data indicate that local therapy of ganirelix would induce tumor reduction in a wide range of uterine leiomyomas.

Stem Cell Growth and Differentiation in Organ Culture: New Insights for Uterine Fibroid Treatment.

Organ culture allows for the understanding of normal and tumor cell biology, and tissues generally remain viable for 5-7 days. Strikingly, we determined that myometrial and MED12 mutant leiomyoma cells repopulated cell-depleted tissue slices after 20 days of culture. Using immunofluorescence and quantitative PCR of stem cell and undifferentiated cell markers, we observed clusters of CD49b+ cells in tumor slices. CD49b+ cells, however, were sparsely detected in the myometrial slices. Almost all LM cells strongly expressed Ki67, while only a few myometrial cells were stained for this proliferation marker. The CD73 marker was expressed only in tumor cells, whereas the mesenchymal stem cell receptor KIT was detected only in normal cells. HMGA2 and CD24 showed broader expression patterns and higher signal intensity in leiomyoma than in myometrial cells. In this study, we propose that activating CD49b+ stem cells in myometrium leads to asymmetrical division, giving rise to transit-amplifying KIT+ cells that differentiate to smooth muscle cells. On the contrary, activated leiomyoma CD49b+ cells symmetrically divide to form clusters of stem cells that divide and differentiate to smooth muscle cells without losing proliferation ability. In conclusion, normal and mutant stem cells can proliferate and differentiate in long-term organ culture, constituting a helpful platform for novel therapeutic discovery.

Dual agonist-antagonist effect of ulipristal acetate in human endometrium and myometrium.

The aim of this study was to assess the molecular effect of ulipristal acetate (UPA) on gene expression in myometrium and endometrium of patients with symptomatic fibroids. Tissues isolated from four women treated preoperatively with UPA (5 mg) were compared to those from untreated controls using NanoString platform to assess the expression of 75 candidate genes modulated by UPA and ovarian steroids. Deregulated genes were then validated by real-time PCR. In myometrium, UPA exerted an antagonistic effect similar to that observed in fibroids. In UPA-treated endometrium, six genes were identified as highly and significantly upregulated, including matricellular genes CCN1 (54-fold, P = 0.0018) and CCN2 (11-fold, P = 0.00044), Krüppel-like factor 4 (>3-fold, P = 0.0036), and mast cell markers including tryptases TPSAB1/TPSB2 (31-fold, P = 0.023) and carboxypeptidase A (CPA3, 17-fold, P = 0.05). In endometrium, UPA induced the expression of genes involved in fibrogenesis and mast cell function-some of them being widely involved in hepatic injury, which could explain the marked fibrosis and inflammatory cell infiltration observed in explanted livers from patients under UPA treatment.

Organotypic culture as a research and preclinical model to study uterine leiomyomas.

Organotypic cultures of tissue slices have been successfully established in lung, prostate, colon, gastric and breast cancer among other malignancies, but until now an ex vivo model based on tissue slices has not been established for uterine leiomyoma. In the present study, we describe a method for culturing tumour slides onto an alginate scaffold. Morphological integrity of tissue slices was maintained for up to 7 days of culture, with cells expressing desmin, estrogen and progesterone receptors. Driver mutations were present in the ex vivo slices at all-time points analyzed. Cultivated tumour slices responded to ovarian hormones stimulation upregulating the expression of genes involved in leiomyoma pathogenesis. This tissue model preserves extracellular matrix, cellular diversity and genetic background simulating more in-vivo-like situations. As a novelty, this platform allows encapsulation of microspheres containing drugs that can be tested on the ex vivo tumour slices. After optimizing drug release rates, microspheres would then be directly tested in animal models through local injection.

HMGA2 and MED12 alterations frequently co-occur in uterine leiomyomas.

OBJECTIVE: Around 70% of uterine leiomyomas show MED12 mutations while overexpression of HMGA2 mRNA is also highly frequent in fibroids. However, previous studies suggested that alterations in both genes are mutually exclusive. In the present study, we searched for mutation in MED12 and analyzed the expression of HMGA2 in 20 uterine leiomyomas and their matched myometrium. METHODS: Normal and tumor tissue obtained from premenopausal women who underwent hysterectomy were collected after surgery and DNA, RNA and proteins were isolated and analyzed for MED12 mutations using Sanger sequencing, HMGA2 mRNA expression by quantitative PCR and HMGA2 protein detection by western blot and immunohistochemistry. RESULTS: 75% of the tumors displayed MED12 mutation while 65% of them showed overexpression of HMGA2 mRNA in leiomyomata compared to myometrial tissues (p = 0,0008). Interestingly, 50% of the tumors showed mutations in MED12 and overexpression of HMGA2 mRNA simultaneously, suggesting that alterations in both genes are relatively frequent in uterine leiomyomas. CONCLUSIONS: Contrary to the present findings, former studies showed that mutations in MED12 and overexpression of HMGA2 are mutually exclusive. Here, we observed that overexpression of HMGA2 mRNA in tumors measured by quantitative PCR and compared to myometrium is a common phenomenon in fibroids and is frequently associated with MED12 mutations. In addition, the common clonal origin of tumors overexpressing HMGA2 mRNA and its expression in few myometrial tissue points to HMGA2 up-regulation as an early event in leiomyoma tumorigenesis.

Altered expression of the tachykinins substance P/neurokinin A/hemokinin-1 and their preferred neurokinin 1/neurokinin 2 receptors in uterine leiomyomata.

OBJECTIVE: To study the expression levels of tachykinins and tachykinin receptors in uterine leiomyomas and matched myometrium. DESIGN: Laboratory study. SETTING: University research laboratories and academic hospital. PATIENT(S): Women undergoing hysterectomy for symptomatic leiomyomas. INTERVENTION(S): Quantitative polymerase chain reaction, immunohistochemistry and Western blot. MAIN OUTCOME MEASURE(S): Expression and tissue immunostaining of substance P, neurokinin A, hemokinin-1, neurokinin 1 receptor full-length (NK1R-Fl) and truncated (NK1R-Tr) isoforms, and neurokinin 2 receptor (NK2R) in paired samples of leiomyoma and adjacent normal myometrium. RESULT(S): TAC1 messenger RNA (mRNA) was significantly up-regulated in leiomyomas, whereas intense immunoreaction for the three peptides was particularly abundant in connective tissue cells. Differential regulation of TACR1 mRNA was observed, and at the protein level there was a significant increased expression of NK1R short isoform (NK1R-Tr). TACR2 mRNA was significantly up-regulated in leiomyomas, although levels of NK2R protein were similar in normal and tumor cells. CONCLUSION(S): These and our previous data demonstrate that the whole tachykinin system is differentially regulated in leiomyomas. The increased expression of NK1R-Tr might stimulate leiomyoma growth in a similar way to that observed in other steroid-dependent tumors.

Expression of Tachykinins and Tachykinin Receptors and Interaction with Kisspeptin in Human Granulosa and Cumulus Cells.

The neurokinin B/NK3 receptor (NK3R) and kisspeptin/kisspeptin receptor (KISS1R), two systems which are essential for reproduction, are coexpressed in human mural granulosa (MGC) and cumulus cells (CCs). However, little is known about the presence of other members of the tachykinin family in the human ovary. In the present study, we analyzed the expression of substance P (SP), hemokinin-1 (HK-1), NK1 receptor (NK1R), and NK2 receptor (NK2R) in MGCs and CCs collected from preovulatory follicles of oocyte donors at the time of oocyte retrieval. RT-PCR, quantitative RT-PCR, immunocytochemistry, and Western blotting were used to investigate the patterns of expression of tachykinin and tachykinin receptor mRNAs and proteins and the possible interaction between the tachykinin family and kisspeptin. Intracellular free Ca(2+) levels ([Ca(2+)]i) in MGCs after exposure to SP or kisspeptin in the presence of SP were also measured. We found that SP, HK-1, the truncated NK1R isoform NK1R-Tr, and NK2R were all expressed in MGCs and CCs. NK1R-Tr mRNA and NK2R mRNA and protein levels were higher in MGCs than in CCs from the same patients. Treatment of cells with kisspeptin modulated the expression of HK-1, NK3R, and KISS1R mRNAs, whereas treatment with SP regulated kisspeptin mRNA levels and reduced the [Ca(2+)]i response produced by kisspeptin. These data demonstrate that the whole tachykinin system is expressed and acts in coordination with kisspeptin to regulate granulosa cell function in the human ovary.

Analysis of the Expression of Tachykinins and Tachykinin Receptors in the Rat Uterus During Early Pregnancy.

The peptides of the tachykinin family participate in the regulation of reproductive function acting at both central and peripheral levels. Our previous data showed that treatment of rats with a tachykinin NK3R antagonist caused a reduction of litter size. In the present study, we analyzed the expression of tachykinins and tachykinin receptors in the rat uterus during early pregnancy. Uterine samples were obtained from early pregnant rats (Days 1-9 of pregnancy) and from nonpregnant rats during the proestrus stage of the ovarian cycle, and real-time quantitative RT-PCR, immunohistochemistry, and Western blot studies were used to investigate the pattern of expression of tachykinins and tachykinin receptors. We found that all tachykinins and tachykinin receptors were locally synthesized in the uterus of early pregnant rats. The expression of substance P, neurokinin B, and the tachykinin receptors NK1R and NK3R mRNAs and proteins underwent major changes during the days around implantation and they were widely distributed in implantation sites, being particularly abundant in decidual cells. These findings support the involvement of the tachykinin system in the series of uterine events that occur around embryo implantation in the rat.

A high-throughput open-array qPCR gene panel to identify housekeeping genes suitable for myometrium and leiomyoma expression analysis.

OBJECTIVE: To evaluate 51 different housekeeping genes for their use as internal standards in myometrial and matched leiomyoma samples in proliferative and secretory phases. METHODS: RNA from 6 myometrium and matched leiomyoma samples was obtained from pre-menopausal women who underwent hysterectomy. Reverse-transcription and real-time quantitative PCR were achieved using TaqMan high-density open-array human endogenous control panel. RESULTS: Expression stability of 51 candidate genes was determined by GeNorm and NormFinder softwares. We identified 10 housekeeping genes, ARF1, MRPL19, FBXW2, PUM1, UBE2D2, EIF2B1, HPRT1, GUSB, ALAS1, and TRIM27, as the best set of normalization genes for comparing relative expression between leiomyoma and myometrium samples in proliferative and secretory phases. CONCLUSIONS: Adequate reference genes for accurate normalization are essential to compare gene expression between leiomyoma and myometrial samples. Ideal housekeeping genes must have stable expression patterns regardless of the sample type and menstrual cycle phase. In this study, we propose a set of 10 candidate genes with greater expression stability than those housekeeping genes commonly used in leiomyoma and myometrium tissues. Their use will improve the sensitivity and specificity of the gene expression analysis in these tissues.

Differentially regulated expression of neurokinin B (NKB)/NK3 receptor system in uterine leiomyomata.

STUDY QUESTION: Are the vasoactive peptide neurokinin B (NKB) and its preferred NK3 receptor (NK3R) differentially expressed in leiomyomas compared with normal myometrium? SUMMARY ANSWER: In leiomyomas, NKB is up-regulated and delocalized, while its preferred NK3R is also differentially regulated. WHAT IS KNOWN ALREADY: The expression of NKB/NK3R in the central nervous system is essential for proper function of the human reproductive axis. Additionally, this system is also widely expressed throughout the female genital tract. Leiomyomas impair fertility and are a major source of abnormal uterine bleeding. The aberrant synthesis of local factors can contribute to the pathological symptoms observed in women with leiomyomata. NKB could be one of these factors, since a vasoactive role of this peptide at a peripheral level has been observed in different systems and species, including humans. NK3R is strongly regulated by estrogens and its activation leads to nuclear translocation affecting chromatin structure and gene expression. STUDY DESIGN, SIZE, DURATION: Samples were obtained between 2006 and 2012 from 28 women of reproductive age at different stages of the menstrual cycle by hysterectomy. Leiomyomas and matched macroscopically normal myometrium from each woman were analysed in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS: RT-PCR, quantitative real time, immunohistochemistry and in situ hybridization were used to investigate the pattern of expression of NKB/NK3R in tissue samples. MAIN RESULTS AND THE ROLE OF CHANCE: Expression of the gene encoding NKB (TAC3) was up-regulated 20-fold in leiomyomas, compared with matched myometrium (P = 0.0008). In tumour tissue, not only connective cells, but also myometrial, endothelial and vascular smooth muscle cells express TAC3 mRNA. Immunoreactivity to NKB was preferentially located in the smooth muscle cell nuclei from normal myometrium in the secretory phase, unlike matched leiomyoma, which showed a predominant cytoplasmic expression pattern. In the normal myometrium, TACR3 mRNA showed variable expression throughout the menstrual phases, with samples showing strong, reduced or no amplification. In leiomyoma, TACR3 was significantly up-regulated compared with matched myometrium (P = 0.0349). LIMITATIONS, REASONS FOR CAUTION: This study is descriptive and although we observed clear differential regulation of the NKB/NK3R system at mRNA and immunohistochemical staining levels in leiomyoma, future functional studies are needed to determine the precise role of NKB in the myometrium in normal and pathological conditions. In addition, further analysis (e.g. in cell culture models) will be required to determine the role of NKB in the nucleus of normal smooth muscle cells, whether nuclear translocation is mediated by NK3R and the consequences of the cytoplasmic expression of NKB in tumour cells. WIDER IMPLICATIONS OF THE FINDINGS: The NKB/NK3R system dysregulation observed in leiomyoma may contribute to the pathological symptoms observed in women with leiomyomata.

Analysis of the expression of neurokinin B, kisspeptin, and their cognate receptors NK3R and KISS1R in the human female genital tract.

OBJECTIVE: To investigate the presence of neurokinin B (NKB)/NK(3) receptor (NK(3)R) and kisspeptin/KISS1 receptor (KISS1R) messenger RNA (mRNA) and proteins throughout the human female genital tract. DESIGN: In vitro study. SETTING: Academic research laboratories and academic hospitals. PATIENT(S): Fifteen reproductive-age women and 16 postmenopausal women provided fresh samples of uterus, ovary, or oviduct, and 12 women provided archival samples of endometrium or oviduct. INTERVENTION(S): Fresh and archival samples of uterus, ovary, and oviduct obtained from reproductive-age and postmenopausal women. MAIN OUTCOME MEASURE(S): Results of reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry to investigate the pattern of expression of NKB/NK(3)R and kisspeptin/KISS1R in target tissues. RESULT(S): Expression of the genes encoding NKB (TAC3) and NK(3)R (TACR3), and kisspeptin (KISS1) and its receptor (KISS1R) was found in the uterus, ovary, and oviduct. Both NKB and NK(3)R immunoreactivity was detected in the endometrium, the oviduct, and the ovary, with marked expression in endometrial and oviductal epithelial cells, where intense coexpression of kisspeptin and KISS1R was also detected. Positive staining for NKB and NK(3)R was found in the myometrium where, in contrast, kisspeptin and KISS1R were absent. CONCLUSION(S): NKB/NK(3)R and kisspeptin/KISS1R are present in female peripheral reproductive tissues with colocalization of both systems in some non-neuronal cell populations of the human female genital tract. Our findings are compatible with a potential modulatory role of NKB and kisspeptin at peripheral reproductive tissues.

Comparative analysis of the ERα/ERß ratio and neurotensin and its high-affinity receptor in myometrium, uterine leiomyoma, atypical leiomyoma, and leiomyosarcoma.

Deregulated steroids are involved in different hormone-dependent tumors, including benign and malignant uterine neoplasms. Leiomyomas (LM) are estrogen and progesterone-dependent benign tumors, whereas "bizarre or atypical LMs" (AL) are considered a subgroup of LM and clinically benign, although their malignant potential is suspect. Uterine leiomyosarcomas (LMS) are malignant smooth muscle tumors, and ovarian steroids may control their growth. Estrogen effects are mediated by 2 receptors, estrogen receptors (ER) α and ß, and the ratio of both receptors seems to be a critical parameter in the estrogen-mediated carcinogenic process. Estradiol induces the expression of neurotensin (NTS), and the coupling of this peptide with its high-affinity receptor, NTS1, has been involved in the regulation of tumoral cell growth. Given the importance of these markers in tumor development, we aim to determine the status of ERα and ERß in the myometrium and LM, AL, and LMS, concomitantly with the expression of NTS/NTS receptor 1 in these tumors. For that purpose, we use immunohistochemistry for all markers analyzed and in-situ hybridization to detect NTS mRNA. These data suggest that LMS are estrogen-dependent tumors, which may use NTS as an autocrine growth factor. In addition, the phenotype of AL with regard to ERα and ERß status and NTS expression is closer to LMS than LM; thus, a potential malignization of this tumor is feasible.

Neurotensin and neurotensin receptor 1 expression in human myometrium and uterine leiomyomas.

Leiomyomas or fibroids are the most frequently diagnosed tumors of the female genital tract, and their growth seems to be steroid-hormone dependent by a yet undetermined cellular and molecular mechanism. Sexual hormones induce the secretion of growth factor peptides and the expression of their receptors, stimulating cell proliferation. One of these factors is neurotensin, and increasing evidence suggests that it can promote growth of different cancer cells. Since there are no data on neurotensin expression in normal and tumoral uterine tissue, we have analyzed the expression of NTS and NTSR1 receptor using immunohistochemistry for protein detection, in situ hybridization to detect cells expressing NTS mRNA, and RT-PCR to detect NTSR1 transcript as well as any of the alternative splice variants recently described for this receptor. We found that NTS and NTSR1 are expressed in connective cells of normal myometrium. In leiomyomas, immunoreactivity for NTS and NTSR1 receptor is colocalized in the smooth muscle cells that are also transcribing NTS. Women receiving high doses of steroids for in vitro fertilization showed tumor growth and increased immunoreactivity for neurotensin and NTSR1 receptor. Interestingly, alternative splice variants of NTSR1 receptor were detected only in tumoral tissue. These findings suggest a role of steroid hormones inducing neurotensin expression in leiomyoma smooth muscle cells. In these cells, NTS could act autocrinally through NTSR1 receptor, promoting their proliferation.

Common variants of the neuropeptide expressing tachykinin genes and susceptibility to asthma: a case-control study.

Since tachykinins appear to be involved in the pathogenesis of allergic asthma, we investigated a possible association between 28 single nucleotide polymorphisms of the tachykinin genes TAC1, TAC3 and TAC4, and asthma susceptibility. A case-control study was conducted on 102 patients and 100 healthy subjects from the Canary Islands (Spain). A significant association with asthma was observed for two SNPs: rs2291855 in the TAC3 gene conferring asthma protection (Odds ratio [OR]: 0.46; 95% Confidence Interval [CI]: 0.22-0.97; P=0.038), and rs4794068 in the TAC4 gene associated with an increased risk for asthma (OR: 1.94; 95% CI: 1.06-3.54; P=0.03). The present study represents a preliminary step in elucidating the association between tachykinin gene polymorphisms and asthma susceptibility.

Differential expression of new splice variants of the neurotensin receptor 1 gene in human prostate cancer cell lines.

Neurotensin is a neuroendocrine peptide acting as a trophic factor in a variety of cells in vivo but it can also function as an autocrine growth factor in human prostate cancer cells in vitro. In addition, the high-affinity G protein-coupled NT receptor (NTS1) is overexpressed in prostate cancer cell lines. Increasing evidence argues for a direct correlation between specific alternative splice variants and cancer. We detected four splice variants of the NTS1 receptor in human prostate cancer cell lines. These isoforms include one or more exons skipping as well as an alternative 5' splice donor site and are expressed in the late-stage androgen independent prostate cancer cell lines PC3 and DU145, but not in the early-stage androgen-sensitive LNCaP or in normal prostate tissue, which only express the normal transcript. This result shows new splice variants of NTS1 for the first time. The differential expression observed among prostate cancer cell lines and normal prostate tissue opens the interesting possibility of a new role of NT/NTS1 pathway in prostate cancer.

Differential expression of neurokinin B and hemokinin-1 in human immune cells.

The tachykinins are a group of related peptides that are mainly synthesized in the central and peripheral nervous system, but are also present in peripheral non-neuronal cells. In humans, substance P (SP) is the most extensively studied tachykinin and is present, along with the NK-1 receptor, in several inflammatory and immune cells. The release of SP under the appropriate stimulus may act as a paracrine or autocrine signal that may help to initiate and/or propagate inflammation. In the present study we have determined the expression pattern of NKB and HK-1 mRNA in human lymphocytes, monocytes, neutrophils and eosinophils. In addition, we have detected for the first time the presence of NKB protein in these cellular types. These findings reinforce the suggestion that tachykinins play a central role in the pathophysiology of the inflammatory process.

mRNA expression of tachykinins and tachykinin receptors in different human tissues.

The tachykinins substance P, neurokinin A and neurokinin B are involved in many pathophysiological processes. A reverse transcription-polymerase chain reaction (RT-PCR) assay was used to analyse the expression of TAC1 and TAC3, the genes that encode substance P/neurokinin A and neurokinin B, respectively, and the genes encoding the tachykinin NK(1), NK(2) and NK(3) receptors in different human tissues. The data show that tachykinins and their receptors mRNAs are broadly distributed in different human tissues being present in neuronal and non-neuronal types of cells. The presence of TAC3 and the tachykinin NK(3) receptor (TACR3) in a wide variety of peripheral tissues argue for a still unexplored role of this ligand-receptor pair in mediating visceral effects of tachykinins. We found, for the first time, that TAC3 and TACR3 mRNAs are expressed in human airways and pulmonary arteries and veins, providing further evidence for the involvement of this system in lung physiopathology.