[Multicenter retrospective study of 38 cases with fumarate hydratase deficiency uterine leiomyoma].

Objective: To investigate the clinicopathological features of fumarate hydratase (FH) deficiency uterine leiomyoma. Methods: The data of 38 patients with FH deficiency uterine leiomyoma were screened and analyzed. The expressions of FH, S-(2-succino)-cysteine (2SC), desmin, p16, p53, CD10 and cell proliferation associated nuclear antigen (Ki-67) proteins were detected by immunohistochemistry, and their clinicopathological features were analyzed retrospectively. Results: (1) Clinical features: the median age of the patients was (42.5±7.4) years old. Twenty-one cases (55%) of them were myomas found in physical examination, and the median maximum diameter of the tumor was 6.0 cm (range: 5.0-7.5 cm); myomectomy was performed in 23 cases (61%), total hysterectomy with or without bilateral appendages in 15 cases (39%); laparoscopic surgery in 27 cases (71%), open surgery in 11 cases (29%); none of the patients had renal cell carcinoma. (2) Histological features: atypical nuclear cells were distributed locally or diffusely, eosinophilic nucleoli and intranuclear inclusion bodies could be seen, glass like globules could be seen in the cytoplasm, nuclear division was 0-4/10 high power field (HPF), and antler like blood vessels and pulmonary edema-like changes could be seen in the stroma. Among 38 patients with FH deficiency uterine leiomyoma, FH was negative in 37 cases (97%), and positive in 1 case (3%); 2SC, desmin, p16, p53, CD10 and Ki-67 showed focal positive expression in 38 cases (100%), including 35 cases (92%) with Ki-67 index<10% and 3 cases (8%) with Ki-67 index ≥10%. (3) Follow-up: 4 cases (11%) recurred, and there was no death. There were significant differences in age, family history, distribution of atypical nuclei and mitosis number between recurrent group and non-recurrent group (all P<0.05). Conclusions: FH deficiency uterine leiomyoma is a rare tumor, which needs pathological examination,immunohistochemical examination and clinical history. Patients younger than 43 years old, with family history, histologically atypical diffuse nuclear distribution and mitotic number ≥3/10 HPF should be alert to the risk of recurrence.

Prevalence of somatic and germline mutations of Fumarate hydratase in uterine leiomyomas from young patients.

AIMS: Hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome is caused by germline mutations in the Fumarate hydratase (FH) gene. In young women, the syndrome often presents with symptomatic uterine leiomyomas, leading to myomectomy or hysterectomy. In this study, we aimed to investigate the incidence and mutational profiles of FH-negative leiomyomas from young patients, thus allowing for early identification and triage of syndromic patients for surveillance. METHODS AND RESULTS: We evaluated 153 cases of uterine leiomyomas from women aged up to 30 years for loss of FH expression by tissue microarray (TMA)-based immunohistochemical staining. Mutational analysis of tumours with loss of FH was carried out by polymerase chain reaction (PCR) amplification of 10 exons within the FH gene and subsequent Sanger sequencing. The status of promoter methylation was assessed by bisulphite sequencing. Loss of FH protein expression was detected in seven (4.6%) of 153 tested uterine leiomyomas from young patients. All FH-negative leiomyomas displayed staghorn vasculature and fibrillary/neurophil-like cytoplasm. We found that six (86%) of seven FH-negative tumours detected by immunohistochemistry harboured FH mutations, 50% of which contained germline mutations. In particular, the germline mutational rate in FH gene was 2.0% (three of 153 cases). Bisulphite sequencing analysis failed to detect promoter methylation in any of the seven tumours. CONCLUSION: Our study showed a relatively high rate of FH germline mutation in FH-negative uterine leiomyomas from patients aged up to 30 years. While genetic mutations confer protein expression loss, epigenetic regulation of the FH gene appears to be unrelated to this phenotype.

Fumarate Hydratase Mutations and Alterations in Leiomyoma With Bizarre Nuclei.

Leiomyoma with bizarre nuclei (LM-BN), is a variant of uterine smooth muscle tumor with atypical histologic features. Although some LM-BN share several significant genetic alterations with leiomyosarcoma, including p16 and p53, the underlying tumorigenesis of LM-BN remains largely unknown. As we previously reported, LM-BN can be divided into 2 subtypes, type I and type II, based on different nuclear features. Type I LM-BN have similar histologic features as uterine smooth muscle tumors with fumarate hydratase (FH) alterations. In this study, we examined FH expression and FH mutations in 77 LM-BN (40 type I cases and 37 type II cases). FH expression was examined by immunohistochemistry using S-(2-succino)-cysteine antibodies (2SC, a protein modification associated with FH inactivation and subsequent fumarate accumulation) and FH antibodies (FH gene products). Seventy-two LM-BN tumors underwent Sanger sequencing to detect FH mutations. We found that 51% (39/77) of LM-BN showed FH alterations detected by immunohistochemistry with both 2SC and FH. Mutational analysis showed that 21% (15/72) of LM-BN harbored FH gene mutations. Further analysis revealed that 85% (34/40) of those with FH alterations were type I LM-BN while 19% (7/37) were type II LM-BN. Our findings suggest that over half of histologically diagnosed LM-BN may be related to FH alterations or FH mutations and the majority of these have the characteristic histologic features of type I LM-BN.

Ovarian steroid hormones modulate the expression of progesterone receptors and histone acetylation patterns in uterine leiomyoma cells.

Uterine leiomyomas are the most common benign smooth muscle cell tumors in women. Estrogen (E2), progesterone (P4) and environmental factors play important roles in the development of these tumors. New treatments, such as mifepristone, have been proposed. We evaluated the gene expression of total (PRT) and B (PRB) progesterone receptors, and the histone acetyltransferase (HAT) and deacetylase (HDAC) activity after treatment with E2, P4 and mifepristone (RU486) in primary cell cultures from uterine leiomyoma and normal myometrium. Compared to myometrium, uterine leiomyoma cells showed an increase in PRT mRNA expression when treated with E2, and increase in PRB mRNA expression when treated with E2 and P4. Treatment with mifepristone had no significant impact on mRNA expression in these cells. The HDAC activity was higher in uterine leiomyoma compared to myometrial cells after treatment with E2 and E2 + P4 + mifepristone. HAT activity was barely detectable. Our results suggest that ovarian steroid hormones modulate PR, and mifepristone was unable to decrease PRT and PRB mRNA. The higher activity of HDAC leiomyoma cells could be involved in transcriptional repression of genes implicated in normal myometrium cell function, contributing to the maintenance and growth of uterine leiomyoma.

MED12 mutations and FH inactivation are mutually exclusive in uterine leiomyomas.

BACKGROUND: Uterine leiomyomas from hereditary leiomyomatosis and renal cell cancer (HLRCC) patients are driven by fumarate hydratase (FH) inactivation or occasionally by mediator complex subunit 12 (MED12) mutations. The aim of this study was to analyse whether MED12 mutations and FH inactivation are mutually exclusive and to determine the contribution of MED12 mutations on HLRCC patients' myomagenesis. METHODS: MED12 exons 1 and 2 mutation screening and 2SC immunohistochemistry indicative for FH deficiency was performed on a comprehensive series of HLRCC patients' (122 specimens) and sporadic (66 specimens) tumours. Gene expression analysis was performed using Affymetrix GeneChip Human Exon Arrays (Affymetrix, Santa Clara, CA, USA). RESULTS: Nine tumours from HLRCC patients harboured a somatic MED12 mutation and were negative for 2SC immunohistochemistry. All remaining successfully analysed lesions (107/116) were deficient for FH. Of sporadic tumours, 35/64 were MED12 mutation positive and none displayed a FH defect. In global gene expression analysis FH-deficient tumours clustered together, whereas HLRCC patients' MED12 mutation-positive tumours clustered together with sporadic MED12 mutation-positive tumours. CONCLUSIONS: Somatic MED12 mutations and biallelic FH inactivation are mutually exclusive in both HLRCC syndrome-associated and sporadic uterine leiomyomas. The great majority of HLRCC patients' uterine leiomyomas are caused by FH inactivation, but incidental tumours driven by somatic MED12 mutations also occur. These MED12 mutation-positive tumours display similar expressional profiles with their sporadic counterparts and are clearly separate from FH-deficient tumours.

[Atypical leiomyoma in a patient with cutaneous leiomyomatosis and mutation of the enzyme fumarate hydratase]. / Leiomioma cutáneo atípico en un paciente con leiomiomatosis cutánea y mutación de la enzima fumarato hidratasa.

We report the case of a 56 year-old male with an atypical leiomyoma in the context of a cutaneous leiomyomatosis and a family history of uterine leiomyomatosis. The genetic study revealed a mutation in the gene for the enzyme fumarate hydratase, but he has not had any renal malignancy so far. Atypical leiomyoma is a rare tumor that usually presents as a single lesion and is exceptional in patients with cutaneous leiomyomatosis. The relation between fumarate hydratase enzyme mutations with multiple leiomyomas, uterine leiomyomatosis and an increased risk of developing kidney cancer is widely known. However, the role of these mutations in the development of atypical leiomyomas is still impossible to clarify given the few cases reported in the literature.

Antioxidant status in women with uterine leiomyoma: relation with sex hormones.

Uterine leiomyomas are benign soft-tissues tumors that arise from uterine smooth muscle tissue. Etiopathogenesis of leiomyomas is not well understood. We aimed to examine whether antioxidant enzyme activities and lipid hydroperoxides level in patients with leiomyoma are influenced by changes in sex hormones and gonadotropins (estradiol (E2), progesterone, FSH, and LH) during menstrual cycle and in postmenopause. The material consisted of blood and uterine tissue specimens. Hormone concentrations were determined and assays for superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activities and lipid hydroperoxides concentration were performed. In blood of examined women, a significant difference in catalase, glutathione peroxidase and glutathione reductase activity was recorded among the phases. There was also a positive correlation between the estradiol/progesterone concentration and the catalase activity. Progesterone negatively correlated with lipid hydroperoxides level. In myoma tissue, we recorded a phase-related difference in lipid hydroperoxides level and activities of superoxide dismutase, glutathione peroxidase activities, and glutathione reductase. Negative correlation was observed between FSH and glutathione peroxidase. The results suggest that antioxidant status in patients with uterine leiomyoma is influenced by the changes in sex hormones during the menstrual cycle and in postmenopause, indicating a role of the observed relationship in the leiomyoma etiology.

Role of single nucleotide polymorphisms in estrogen-metabolizing enzymes and susceptibility to uterine leiomyoma in Han Chinese: a case-control study.

AIM: To explore the relationship between estrogen metabolism enzyme gene polymorphism and susceptibility to uterine fibroids, and to seek the screening molecular markers for genetic traits in uterine fibroid populations. METHODS: A total of 300 female Han Chinese patients and 300 healthy female Han Chinese volunteers in Nanjing (age range, 30-50 years) were recruited from Zhongda Hospital, Southeast University from February 2011 to March 2012. The single nucleotide polymorphisms (SNP) of estrogen-metabolizing enzyme genes from the two groups of women were examined by polymerase chain reaction denaturing high-performance liquid chromatography, which were four COMT gene loci including rs3087869, rs165774, rs165599 and rs4680, three CYP1A1 gene loci including rs1048943, rs4646421 and rs4646422, and three CYP1B1 gene loci including rs1056827, rs1056836 and rs1056837. Genotype frequencies among cases and controls were calculated and analyzed by binary logistic regression. RESULTS: Regression analysis of SNP showed that COMT IVS1+2329C>T (odds ratio [OR], 2.872; 95% CI, 1.690-4.882) and Val158Met (OR, 2.593; 95% CI, 1.546-4.350), CYP1A1 Ile462Val (OR, 2.383; 95% CI, 1.418-4.005) and Gly45Asp (OR, 2.489; 95% CI, 1.49-4.159), and CYP1B1 Ala119Ser (OR, 3.361; 95% CI, 2.035-5.552) and Leu432Val (OR, 0.164; 95% CI, 0.061-0.441) influenced uterine fibroids significantly (P < 0.05). Allele and genotype frequencies among cases and control were calculated and examined to match the Hardy-Weinberg equilibrium with the χ²-test. CONCLUSION: The genetic polymorphisms of IVS1+2329C>T and Val158Met loci in COMT, Ile462Val and Gly45Asp loci in CYP1A1 and Ala119Ser loci in CYP1B1 were risk factors for uterine leiomyoma development, and Leu432Val locus in CYB1B1 may be a protective factor. The results provide a theoretical basis for genetic screening and early intervention for uterine leiomyoma-susceptible populations.

Vitamin D3 inhibits expression and activities of matrix metalloproteinase-2 and -9 in human uterine fibroid cells.

STUDY QUESTION: Can biologically active vitamin D3 [1,25(OH)2D3] regulate the expression and activity of matrix metalloproteinases (MMPs) in human uterine fibroid cells? SUMMARY ANSWER: 1,25(OH)2D3 effectively reduced the expression and activities of MMP-2 and MMP-9 in cultured human uterine fibroid cells. WHAT IS KNOWN ALREADY: Uterine fibroids (leiomyoma) express higher levels of MMP activity than adjacent normal myometrium, and this is associated with uterine fibroid pathogenesis. However, it is unknown whether 1,25(OH)2D3 can regulate the expression and activities of MMPs in human uterine fibroid cells. STUDY DESIGN, SIZE, DURATION: Surgically removed fresh fibroid tissue was used to generate primary uterine fibroid cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: An immortalized human uterine fibroid cell line (HuLM) and/or primary human uterine fibroid cells isolated from fresh fibroid tissue were used to examine the expression of several MMPs, tissue inhibitors of metalloproteinases (TIMP) 1 and 2 and the activities of MMP-2 and MMP-9 after 1,25(OH)2D3 treatment. Real-time PCR and western blots analyses were used to measure mRNA and protein expression of MMPs, respectively. Supernatant cell culture media were analyzed for MMP-2 and MMP-9 activities using a gelatin zymography assay. MAIN RESULTS AND THE ROLE OF CHANCE: 1-1000 nM 1,25(OH)2D3 significantly reduced mRNA levels of MMP-2 and MMP-9 in HuLM cells in a concentration-dependent manner (P < 0.5 to P < 0.001). The mRNA levels of MMP-1, MMP-3, MMP-13 and MMP-14 in HuLM cells were also reduced by 1,25(OH)2D3. 1,25(OH)2D3 significantly reduced MMP-2 and MMP-9 protein levels in a concentration-dependent manner in both HuLM and primary uterine fibroid cells (P < 0.05 to P < 0.001). Moreover, 1,25(OH)2D3 increased the mRNA levels of vitamin D receptor (VDR) and TIMP-2 in a concentration-dependent manner in HuLM cells (P < 0.05 to P < 0.01). 1,25(OH)2D3 also significantly increased protein levels of VDR and TIMP-2 in all cell types tested (P < 0.05 to P < 0.001). Gelatin zymography revealed that pro-MMP-2, active MMP-2 and pro-MMP-9 were down-regulated by 1,25(OH)2D3 in a concentration-dependent manner; however, the active MMP-9 was undetectable. LIMITATIONS, REASONS FOR CAUTION: This study was performed using in vitro uterine fibroid cell cultures and the results were extrapolated to in vivo situation of uterine fibroids. Moreover, in this study the interaction of vitamin D3 with other regulators such as steroid hormone receptors was not explored. WIDER IMPLICATIONS OF THE FINDINGS: This study reveals an important biological function of 1,25(OH)2D3 in the regulation of expression and activities of MMP-2 and MMP-9. Thus, 1,25(OH)2D3 might be a potential effective, safe non-surgical treatment option for human uterine fibroids.

Polymorphism of catechol-o-methyltransferase and uterine leiomyoma.

Uterine leiomyoma (ULM) is the most common gynecological benign tumor that is affecting around 20-50 % of women over the age of 30. Although its molecular pathogenesis is still unknown, ULM has a multifactorial etiology determined by both genetics and environmental factors. The present study was designed to find out whether Val158Met polymorphism in the catechol-o-methyltransferase (COMT) gene is associated with the risk of ULM. We analyzed COMT Val158Met polymorphism in 105 ULMs patients and 105 healthy subjects using a polymerase chain reaction-based restriction fragment length polymorphism assay. We found remarkably similar frequencies in ULM compared with controls for COMT Val158Met genotypes and alleles, and no association was found between ULM and this polymorphism (p = 0.46). The COMT 158 Met allele in patients with large (≥5 cm) fibroids was higher than in patients with small (<5 cm) fibroids, and significant association was found between fibroid size and COMT 158 Met allele (p = 0.011, OR 0.50, 95 %CI 0.28-0.90). Our results reflect that COMT Val158Met polymorphism is not associated with an increased risk of ULMs, but Val158Met polymorphism may be a risk factor for development of large fibroids in Turkish patients with ULM.

Bilateral areolar leiomyomas in a patient undergoing BRAF inhibition therapy for melanoma.

BRAF inhibition therapy, used to treat melanomas with BRAF mutations, is associated with both neoplastic and non-neoplastic cutaneous side effects including squamous cell carcinomas, warty dyskeratomas, verrucous keratoses, photosensitivity and widespread eruptions that present histopathologically as acantholytic dyskeratosis. We report a case of a patient undergoing BRAF inhibition therapy for disseminated melanoma with a V600E mutation who developed bilateral areolar leiomyomas, one of which was biopsied and the other of which resolved after discontinuation of vemurafenib therapy. To our knowledge, this is the first reported case of a mesenchymal neoplasm developing in association with BRAF inhibition therapy.

Fumarate hydratase gene mutation in two young patients with sporadic uterine fibroids.

Fumarate hydratase (FH) is a key enzyme of the Krebs cycle. Germline mutations in the FH gene encoding fumarate hydratase cause autosomal dominant syndromes multiple cutaneous and uterine leiomyomata and hereditary leiomyomatosis and renal cell cancer (HLRCC). Few data have been published on the role of FH gene mutation in development of uterine fibroids outside the context of multiple cutaneous and uterine leiomyomata /HLRCC. We report two FH gene mutations, one novel and one previously described, in two young patients with sporadic uterine fibroids and decreased fumarate hydratase activity in lymphocytes. In patient 1, a novel heterozygous mutation c.892G>C was found. In patient 2 we detected heterozygous mutation c.584T>C. Both the patients had a negative family history for renal cancer and cutaneous leiomyomatosis. None of the relatives, however, underwent renal imaging at the time of writing. FH mutation carriers may be easily identified by analysis of fumarate hydratase activity in blood lymphocytes. We suggest performing fumarate hydratase activity or FH mutation screening in women with onset of uterine fibroids in their 20s and family history of uterine fibroids or other HLRCC-associated malignancies.

Expression of endothelial nitric oxide synthase in the uterus of patients with leiomyoma or adenomyosis.

AIM: To confirm the difference in the expression of endothelial nitric oxide synthase in the normal endometrium and myometrium of women who have leiomyoma or adenomyosis compared with controls, and its correlation with the pathogenesis of menorrhagia or dysmenorrhea in patients with uterine leiomyoma. METHODS: Fifty-one hysterectomized patients were divided into three groups: (i) patients with leiomyoma (n=24); (ii) those with adenomyosis (n = 19); and (iii) the control group (n=8). The expression of endothelial nitric oxide synthase was confirmed on immunohistochemistry and analyzed using an evaluation nomogram. RESULTS: The expression of endothelial nitric oxide synthase was significantly higher in the leiomyoma group and the adenomyosis group as compared with the control group. In the subgroup analysis of leiomyoma depending on symptoms (menorrhagia or dysmenorrhea or both), the expression of endothelial nitric oxide synthase was significantly higher in the symptomatic subgroup than the asymptomatic subgroup (endometrium P=0.0029, myometrium P=0.0276). CONCLUSIONS: Based on the findings that the expression of endothelial nitric oxide synthase was significantly higher in the uterus with leiomyoma or adenomyosis, it can therefore be inferred that nitric oxide might have a pathological effect on the uterus with the above diseases. In particular, it is also presumed that endothelial nitric oxide synthase is closely associated with menorrhagia and dysmenorrhea.

Human prolyl hydroxylase expression in uterine leiomyoma during the menstrual cycle.

BACKGROUND: To investigate the role of prolyl hydroxylase (PH), a key enzyme of collagen synthesis, in human uterine leiomyoma, PH expression was determined in the normal uterine myometrium and the leiomyoma tissues during the menstrual cycle. METHODS: The tissues were obtained from 40 regularly cycling women (aged 29 to 53 yr) who were undergoing abdominal hysterectomy for symptomatic uterine leiomyoma. Immunohistochemistry for human PH with specific monoclonal antibody was used for analysis. RESULTS: Immunohistochemical staining for PH revealed intense staining of leiomyoma cells in the uterine leiomyoma throughout the menstrual cycle, as compared with the adjacent normal myometrium. In the secretory phase, weak or no immunostaining for PH was detected in the normal myometrial tissues. CONCLUSIONS: These results suggest that increased expression of PH might play an role in the physiology of uterine leiomyoma during the menstrual cycle.

Oxidative stress and prolidase activity in women with uterine fibroids.

The aim of this study was to investigate oxidative stress markers and prolidase activity in serum and tissue samples of women with uterine fibroids, with further analysis on position and size. Lipid hydroperoxide, ceruloplasmin, catalase, arylesterase, free sulfhydryl group activity and prolidase activity levels were measured in fibroid tissue, myometrial tissue and serum of the same patients (n = 51), at the same time. Results show that ceruloplasmin, catalase, arylesterase, free sulfhydryl group and prolidase activities were higher in fibroid tissue than those in myometrial tissue (p = 0.003, 0.009, 0.004, 0.02, 0.008, respectively). Serum levels of catalase and prolidase were lower, and arylesterase and free sulfhydryl groups were higher in the fibroid group than those in the control group (p < 0.001 for all). Fibroid volume in submucosal subgroup of the fibroid group yield significant correlation with ceruloplasmin, catalase, arylesterase and prolidase activities (r = 0.84, p = 0.02; r = 0.93, p < 0.001; r = 0.63, p = 0.049 and r = 0.87, p = 0.01, respectively). Despite the lack of statistical significance, the highest levels of prolidase activity were found in fibroid samples, especially in submucosal ones. It is concluded that this study demonstrated increased antioxidative repair system in the fibroid tissue compared to the myometrium and serum of the same patients. Additionally, higher pathophysiological potential of the submucosal fibroids over intramural and subserosal fibroids were shown with the levels of oxidative stress markers and prolidase activity levels.

[Association of polymorph variants of CYP1A2 and CYP1A1 genes with reproductive and thyroid diseases in female workers of petrochemical industry].

The article presents results obtained in study of relationship between polymorph variants of CYP1A1 and CYP1A2 genes with reproductive and thyroid diseases risk in female workers of petrochemical industry, when compared with reference group females. Variants TD and DD of CYP1A2 gene appeared to be associated with nodes formation in uterus and breast in female workers and reference group females. Following liability markers are obtained: homozygous in rare allele genotype CC of CYP1A1 gene for reproductive and thyroid diseaes (fibrous cystic mastopathy and nodular goitre), heterozygous genotype AG of CYP1A1 gene in uterine myoma and fibrous cystic mastopathy, homozygous in deleted T genotype of CYP1A2 gene in autoimmune thyroiditis. Occupational hazards and long length of service at hazardous industries increase effects of rare alleles of the genes studied.

Tryptophan 2,3-Dioxygenase-2 in Uterine Leiomyoma: Dysregulation by MED12 Mutation Status.

Uterine leiomyomas (fibroids) are common benign tumors in women. The tryptophan metabolism through the kynurenine pathway plays important roles in tumorigenesis in general. Leiomyomas expressing mutated mediator complex subunit 12 (mut-MED12) were reported to contain significantly decreased tryptophan levels; the underlying mechanism and the role of the tryptophan metabolism-kynurenine pathway in leiomyoma tumorigenesis, however, remain unknown. We here assessed the expression and regulation of the key enzymes that metabolize tryptophan. Among these, the tissue mRNA levels of tryptophan 2,3-dioxygenase (TDO2), the rate limiting enzyme of tryptophan metabolism through the kynurenine pathway, was 36-fold higher in mut-MED12 compared to adjacent myometrium (P < 0.0001), and 14-fold higher compared to wild type (wt)-MED12 leiomyoma (P < 0.05). The mRNA levels of other tryptophan metabolizing enzymes, IDO1 and IDO2, were low and not significantly different, suggesting that TDO2 is the key enzyme responsible for reduced tryptophan levels in mut-MED12 leiomyoma. R5020 and medroxyprogesterone acetate (MPA), two progesterone agonists, regulated TDO2 gene expression in primary myometrial and leiomyoma cells expressing wt-MED12; however, this effect was absent or blunted in leiomyoma cells expressing G44D mut-MED12. These data suggest that MED12 mutation may alter progesterone-mediated TDO2 expression in leiomyoma, leading to lower levels of tryptophan in mut-MED12 leiomyoma. This highlights that fibroids can vary widely in their response to progesterone as a result of mutation status and provides some insight for understanding the effect of tryptophan-kynurenine pathway on leiomyoma tumorigenesis and identifying targeted interventions for fibroids based on their distinct molecular signatures.

Histone deacetylase inhibition by suberoylanilide hydroxamic acid: a therapeutic approach to treat human uterine leiomyoma.

OBJECTIVE: To evaluate the effect of inhibition of histone deacetylases (HDACs) by suberoylanilide hydroxamic acid (SAHA) treatment of human uterine leiomyoma primary (HULP) cells in vitro on cell proliferation, cell cycle, extracellular matrix (ECM) formation, and transforming growth factor ß3 (TGF-ß3) signaling. DESIGN: Prospective study comparing uterine leiomyoma (UL) vs. adjacent myometrium (MM) tissue and cells with or without SAHA treatment. SETTING: Hospital and university laboratories. PATIENT(S): Women with UL without any hormone treatment. INTERVENTION(S): Myomectomy or hysterectomy surgery in women for leiomyoma disease. MAIN OUTCOME MEASURE(S): HDAC activity was assessed by enzyme-linked immunosorbent assay, and gene expression was assessed by quantitative real-time polymerase chain reaction. Effects of SAHA on HULP cells were analyzed by CellTiter (Promega, Madison, Wisconsin), Western blot, and quantitative real-time polymerase chain reaction. RESULT(S): The expression of HDAC genes (HDAC1, fold change [FC] = 1.65; HDAC3, FC = 2.08; HDAC6, FC = 2.42) and activity (0.56 vs. 0.10 optical density [OD]/h/mg) was significantly increased in UL vs. MM tissue. SAHA decreased HDAC activity in HULP cells but not in MM cells. Cell viability significantly decreased in HULP cells (81.68% at 5 µM SAHA, 73.46% at 10 µM SAHA), but not in MM cells. Proliferating cell nuclear antigen expression was significantly inhibited in SAHA-treated HULP cells (5 µM SAHA, FC = 0.556; 10 µM SAHA, FC = 0.622). Cell cycle markers, including C-MYC (5 µM SAHA, FC = 0.828) and CCND1 (5 µM SAHA, FC = 0.583; 10 µM SAHA, FC = 0.482), were significantly down-regulated after SAHA treatment. SAHA significantly inhibited ECM protein expression, including FIBRONECTIN (5 µM SAHA, FC = 0.815; 10 µM SAHA, FC = 0.673) and COLLAGEN I (5 µM SAHA, FC = 0.599; 10 µM SAHA, FC = 0.635), in HULP cells. TGFß3 and MMP9 gene expression was also significantly down-regulated by 10 µM SAHA (TGFß3, FC = 0.596; MMP9, FC = 0.677). CONCLUSION(S): SAHA treatment inhibits cell proliferation, cell cycle, ECM formation, and TGF-ß3 signaling in HULP cells, suggesting that histone deacetylation may be useful for treatment of UL.

Hypermethylated DAPK in serum DNA of women with uterine leiomyoma is a biomarker not restricted to cancer.

OBJECTIVE: Ovarian cancer is most frequently diagnosed at a late stage with a poor prognosis. No markers for early diagnosis have been established. Aberrantly methylated DNA appears as a promising molecular cancer marker. The aim of this study was to analyze the methylation status of the proapoptotic cancer related gene death-associated protein kinase (DAPK) in ovarian cancer patients, healthy controls and in patients suffering from a benign proliferative disease such as uterine leiomyoma. METHODS: Methylation-specific PCR (MSP) was used to detect DAPK methylation in primary tumor tissue and serum of both ovarian cancer (n=32) and uterine leiomyoma patients (n=17 primary tissue, n=30 serum). Serum samples from healthy women served as controls (n=20). MSP results were confirmed by restriction digest and sequencing analyses of cloned PCR products. RESULTS: DAPK methylation was detected in 50% and 35.3% of primary tissue and 56% and 23.8% of serum samples from ovarian cancer and leiomyoma patients, respectively. However, the association of methylation frequencies in tissue and serum was low (kappa=-0.053). Sequencing experiments revealed fully methylated MSP products in sera of both ovarian cancer and leiomyoma patients. In contrast sera from control patients showed only partially methylated DAPK sequences. CONCLUSION: DAPK hypermethylation was neither specific for the tissue of origin nor for cancer. The high prevalence of leiomyoma compromises the utility of this gene as a serum marker for early ovarian cancer detection. These data emphasize the necessity to co-analyze controls presenting with non-cancer proliferative disease in the quest for molecular cancer markers.

Tryptophan catabolism is dysregulated in leiomyomas.

OBJECTIVE: To determine the expression and functional roles of indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO2) in leiomyoma. DESIGN: Experimental study. SETTING: Academic research laboratory. PATIENT(S): Women undergoing hysterectomy for leiomyoma. INTERVENTION(S): Blockade of IDO1 and TDO2. MAIN OUTCOME MEASURE(S): Expression of IDO1 and TDO2 in leiomyoma and the effects of their inhibitors on the extracellular matrix. RESULT(S): Leiomyoma expressed significantly higher levels of IDO1 and TDO2 messenger ribonucleic acid (mRNA; 60.3%, 35/58 pairs and 98.3%, 57/58 pairs, respectively) and protein (54%, 27/50 pairs and 92%, 46/50 pairs, respectively) as well as the enzyme activity marker kynurenine (78.3%, 36/46 pairs for IDO1/TDO2) compared with levels in matched myometrium. The expression of TDO2 but not IDO1 mRNA was significantly higher in fibroids from African American compared with that in Caucasian and Hispanic patients. The TDO2 but not the IDO1 protein and mRNA levels were more abundant in fibroids bearing the MED12 mutation compared with results in wild-type leiomyomas. Treatment of leiomyoma smooth muscle cell and myometrial smooth muscle cell spheroids with the TDO2 inhibitor 680C91 but not the IDO1 inhibitor epacadostat significantly repressed cell proliferation and the expression of collagen type I (COL1A1) and type III (COL3A1) in a dose-dependent manner; these effects were more pronounced in leiomyoma smooth muscle cells compared with myometrial smooth muscle cell spheroids. CONCLUSION(S): These results underscore the physiological significance of the tryptophan degradation pathway in the pathogenesis of leiomyomas and the potential utility of anti-TDO2 drugs for treatment of leiomyomas.