Leiomyoma Cells Research Articles

NAV3, a Tumor Suppressor Gene, Is Decreased in Uterine Leiomyoma Tissue and Cells.

NAV 3 is a tumor suppressor of unknown function in leiomyomas. The objective of this study is to assess NAV3 expression and its potential role in human uterine leiomyomas. NAV3 protein expression was examined in patient leiomyoma and patient-matched myometrial tissue samples by Western blot and immunohistochemistry. NAV3 mRNA and protein expression was assessed in leuprolide acetate- and cetrorelix-treated cell line leiomyoma samples. RNAseq analysis of placebo-treated leiomyoma compared with myometrium demonstrated the presence of transcripts encoding for several neuronal proteins. For NAV3, RNA sequence analysis demonstrated decreased expression in leiomyoma as compared with myometrium (0.86 ± 0.03 fold). Presence of NAV3 mRNA was also decreased in leiomyoma surgical samples (0.43 fold ± 0.05, p = 0.026) compared with patient-matched myometrium. Confirmatory qRT-PCR results on immortalized leiomyoma and myometrial cell lines similarly demonstrated a decrease in expression of NAV3 in leiomyomas (0.28 ± 0.02, p = 0.00075). Immunohistochemical analysis demonstrated a significant decrease in NAV 3 protein in leiomyomas (H-score 154.7 ± 6.2) as compared with myometrium (H-score; 312.5 ± 14.7, p < 0.0001). Leuprolide acetate-treated leiomyoma cells demonstrated an increase in NAV 3 mRNA expression (1.53 ± 0.13, p < 0.0001). Similarly, Western blot analysis on leuprolide-treated leiomyoma cells showed a non-significant increase in NAV 3 protein expression (1.26 ± 0.09, p = 0.063). NAV 3, a tumor suppressor in numerous cancers, is decreased in leiomyoma cells and tissue compared with myometrium, and increased by GnRH analog treatment, suggesting that NAV3 may mediate steroid hormone-independent leiomyoma regulation by GnRH analogs.

Steroid hormones and hormone antagonists regulate the neural marker neurotrimin in uterine leiomyoma

To characterize the role of steroid hormone and antihormone exposure on neurotrimin (NTM) expression in human leiomyoma and myometrial tissue and cells. Laboratory study of placebo and ulipristal acetate (UPA)-treated patient tissue. Invitro assessment of immortalized myometrial and leiomyoma cell lines after hormone and antihormone exposure. Academic research center. Not applicable. Exposure of leiomyoma cell lines to 17β-E2, medroxyprogesterone acetate (MPA), UPA, and fulvestrant. Messenger RNA expression quantified with the use of RNASeq analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Protein levels quantified by means of Western blot analysis. Immunohistochemistry (IHC) on placebo- and UPA-treated patient uterine tissue specimens. Expression of NTM in human uterine leiomyoma specimens according to RNASeq was increased compared with myometrium (5.22 ± 0.57-fold), which was confirmed with the use of qRT-PCR (1.95 ± 0.05). Furthermore, NTM protein was elevated in leiomyoma tissue compared with matched myometrium (2.799 ± 0.575). IHC revealed increased staining intensity in leiomyoma surgical specimens compared with matched myometrium of placebo patients. Western blot analysis in immortalized leiomyoma cell lines demonstrated an up-regulation of NTM protein expression (2.4 ± 0.04). Treatment of leiomyoma cell lines with 17β-E2 yielded a 1.98 ± 0.11-fold increase in NTM protein expression; however, treatment with fulvestrant showed no significant change compared with control. Leiomyoma cell lines demonstrated a 1.91 ± 0.97-fold increase in NTM protein expression after progesterone treatment. RNASeq analysis demonstrated a reduced expression in patient leiomyoma after UPA treatment (0.75 ± 0.14). Treatment of leiomyoma cells with UPA demonstrated a reduced total NTM protein amount (0.54 ± 0.31) in patients, which was confirmed with the use of IHC (UPA10 147.2 ± 9.40, UPA20 182.8 ± 8.98). Invitro studies with UPA treatment revealed a concentration-dependent effect that supported these findings. NTM, a neural cell adhesion molecule, is increased in leiomyoma compared with myometrium in patient tissue and invitro models after estrogen and progesterone treatment. Down-regulation of expression occurs after UPA treatment, but not after fulvestrant exposure. NCT00290251.

The possible involvement of interleukin-6 in congestive heart failure in a postmenopausal woman with uterine fibromatosis

Interleukin-6 (IL-6) has been shown to contribute to the progression of myocardial damage and dysfunction in congestive heart failure. We report a case of a 53-year old woman with congestive heart failure that may have been caused by IL-6 secreting uterine leiomyoma. The woman developed progressive dyspnea, pleural effusion, and leg edema with a decreased left ventricular ejection fraction (LVEF). Imaging studies indicated uterine leiomyoma compressing the vena cava. Due to unresponsiveness to conventional medical treatment, she underwent a total abdominal hysterectomy with bilateral salpingo-oophorectomy to alleviate compression of the vena cava. After surgery, pleural effusion and leg edema completely resolved. Concomitant with the subsequent improvement of LVEF, a pre-operative serum IL-6 measurement of 36.2 pg/mL fell to 1.6 pg/mL postoperatively. Immunologically positive staining for IL-6 was demonstrated in the cytoplasm of most of the uterine leiomyoma cells. These findings suggest that IL-6 produced by leiomyoma cells may have been involved in the pathophysiological progression of heart failure and associated hemodynamic changes.

CORRIGENDUM FOR "Role of Activin-A and Myostatin and Their Signaling Pathway in Human Myometrial and Leiomyoma Cell Function".

CORRIGENDUM FOR "Role of Activin-A and Myostatin and Their Signaling Pathway in Human Myometrial and Leiomyoma Cell Function".

Autophagy up-regulation by ulipristal acetate as a novel target mechanism in the treatment of uterine leiomyoma: an in vitro study

To assess the effect of ulipristal acetate (UPA) on the autophagic process of uterine leiomyoma cells. Invitro study in primary cultures of leiomyoma and myometrial cells isolated from biopsy specimen, and gene expression evaluation in biopsy material. Cellular pathology laboratory. Premenopausal women (without hormonal treatment) undergoing myomectomy or hysterectomy for symptomatic leiomyomas. Surgical specimens collected from uterine leiomyomas and matched normal myometria. After treatment of myometrial and leiomyoma cells with UPA, autophagy was evaluated by Western blot analysis of the typical biochemical markers, LC3-II, LC3-II:LC3-I ratio, and p62/SQSTM1. The expression level of Atg7 and Atg4D proteins was also assessed by Western blot. The increase of LC3-II protein, LC3-II:LC3-I ratio, and p62/SQSTM1 protein indicates that UPA treatment up-regulates the autophagic response in leiomyoma cells, whereas these markers were almost unchanged in myometrial cells. Consistently, an increased level of Atg7 and Atg4D proteins was observed only in UPA-treated leiomyoma cells. The autophagic machinery is put into motion selectively in these cells, despite that the basal messenger RNA levels of LC3, SQSTM1, and ATG7 in leiomyoma biopsy specimen were not significantly different from those found in normal myometrial biopsy material. Invitro UPA treatment stimulates the autophagic response selectively in leiomyoma cells, which adds a novel indication for the clinical use of this selective P receptor (PR) modulator. Autophagy up-regulation may potentially contribute to the leiomyoma shrinkage occurring in UPA-treated patients and warrants further study.

Melatonin activates cell death programs for the suppression of uterine leiomyoma cell proliferation.

The circadian nature of melatonin has a protective effect on the progression of female reproductive cancers, including breast and ovarian cancers. However, the effect of melatonin on the growth of uterine leiomyoma is still unclear. In this study, we found that the growth of uterine leiomyoma ELT3 cells was reduced by treatment with melatonin. Treatment with melatonin increased the distribution of sub-G1 phase and increased DNA condensation in ELT3 cells. Melatonin-induced apoptosis and autophagy cell death progression were observed in ELT3 cells. Melatonin exerts a highly selective effect on primary normal human uterine smooth muscle (UtSMC) cells. The UtSMC cell cycle was arrested by melatonin treatment through up-regulation of p21, p27, and PTEN protein expression, but melatonin did not further promote apoptosis program activation. Melatonin reduced cell proliferation in ELT3 cells underlying the activation of melatonin MT1 and MT2 receptors, which in turn down-regulated the Akt-ERK1/2-NFκB signaling pathway. Melatonin reduced ELT3 tumor growth in both xenograft and orthotopic uterine tumor mice models. The extracellular matrix of the tumor was also reduced by melatonin treatment. Taken together, these results suggest that melatonin potentially plays a role in suppression of uterine leiomyoma growth.

MiR-93 blocks cell cycle progression and promotes apoptosis in uterine leiomyoma cells by targeting CCND1.

Uterine leiomyoma (UL) is the most common type of benign tumor in the women's reproductive system. A number of genes has been found to play an important role in the initiation and progression of UL, including miRNAs. In this study, our results exhibited that miR-93, a member of mir-106b-25 cluster, significantly reduced the cell viability, promoted cell cycle arrest, caused apoptosis, and inhibited migration in UL cells (p < .01). Moreover, our results have provided experimental evidence that miR-93 regulated the biological functions of UL cells by targeting CCND1.

Effects of progesterone receptor on proliferation of uterine leiomyoma cells.

In order to study the mechanism of the effect of progesterone receptor on the growth of primary uterine leiomyoma cells, the primary cells were extracted from uterine leiomyoma cells and identified by immunohistochemistry (IHC). Mitochondrial progesterone receptor-positive [PR-M(+)], mitochondrial progesterone receptor-negative [PR-M(-)], progesterone receptor A (PR-A) and progesterone receptor B (PR-B) were screened by Western blotting. Different concentrations of Mifepristone (MIF), a progesterone receptor antagonist, were used to interfere with PR-M(+) and PR-M(-) cell lines, respectively. Proliferation and apoptosis of PR-M(+) and PR-M(-) cell lines were detected by tetramethylazolyl blue method and flow cytometry, respectively. The expression of Caspase-3 and B-cell lymphoma 2 (Bcl-2) protein was detected by Western blotting. The results showed that the growth of PR-M(+) and PR-M(-) uterine leiomyoma cells was inhibited with the increase of MIF concentration. Furthermore, the proliferation inhibition rate and apoptosis rate were gradually increased. However, the expression of Caspase-3 protein on progesterone receptor M increased, while the expression of Bcl-2 decreased. Moreover, progesterone could induce progesterone receptor M to up-regulate apoptotic protein Caspase-3 and down-regulate anti-apoptotic protein Bcl-2, thus it could inhibit the apoptosis of primary cultured uterine leiomyoma cells and promote the proliferation of leiomyoma cells.

Isoliquiritigenin induces cell death programs and reduces ECM accumulation in uterine leiomyoma

Abstract Background Uterine leiomyomas, also known as fibroids, are the comment and high prevalence in women of reproductive age. Isoliquiritigenin (ISL), a licorice flavonoid, exerts variant biological properties. In this study, the effect of ISL on anti-proliferation of uterine leiomyoma was investigated. Methods Cell viability was determined using the MTT assay. The protein expression was assayed using Western blot analysis. Cell apoptosis was assayed using Annexin V-FITC/PI and Hoechst 33342 staining. Results The results showed that treatment of uterine leiomyoma ELT3 cells and primary uterine smooth muscle UtSMC cells with ISL alone or ISL plus estradiol (E2) reduced cell proliferation. Cotreatment with E2 and ISL inhibit G2/M and S phase arrest in ELT3 and UtSMC cells, respectively. The higher sub-G1 phase arrest and fluorescence intensity of Hoechst 33342 nucleus stain were observed in cotreatmenet of ELT3 cells with E2 and ISL compared with UtSMC cells. Furthermore, cotreated ELT3 cells with E2 and ISL induced apoptosis and autophagy cell death programs. The E2-enhanced mice uterine myometrium growth was reduced by ISL presence, resulting in reduced the myometrium layer. Moreover, ISL reduced extracellular matrix proteins and matrix metalloproteinase (MMPs) expression, whereas increased tissue inhibitor of MMPs (TIMPs) expression. Conclusion Our findings indicated the inhibitory effects of ISL on the animal model and indicated that ISL reduced ECM-related protein expression and cell proliferation in the leiomyoma cell line. Taken together, our results suggested that ISL could be considered as a new option for the treatment of uterine leiomyoma.

IL6 and STAT-3 pathway highlight the differences in molecular responses in myometrium and uterine fibroids

Human fibroids are highly prevalent and symptomatic uterine tumors. Phenotypically, they are different from normal myometrium because of massive production of extracellular matrix (ECM), which is a hallmark of these benign tumors resulting in symptoms including abnormal bleeding and pain. Dysregulated production of inflammatory cytokine, IL6 and its regulated JAK/STAT-3 pathway are known to contribute to the fibrotic process. Our objective was to understand the effect of IL6 on fibroid and myometrium cells and the role of JAK/STAT pathway in production of matrix proteins that play a major role in fibroid pathogenesis. Laboratory study. Leiomyoma and patient-matched myometrium cell lines exposed to different concentrations of IL6 and JAK/STAT-3 modulators for various exposure time periods, in a 2D culture model system. Changes in expression of ECM proteins and regulating pathways were assessed using western blot. In leiomyoma cells, IL6 activation of STAT-3 peaked (2.32+/- 0.21 fold) as early as 1.5hr of continuous exposure, and was 1.4+/- 0.04 fold increased at end of 3hr of exposure. The maximum increase (1.59+/- 0.07 fold) in the feedback loop protein, Suppressor of Cytokine Signaling (SOCS3) required a short exposure to IL6 (30min) followed by collection after 3hr. We also observed an increase (1.9+/- 0.11 fold) in the ECM structural collagen-1 after continuous exposure to IL6 (3hr) in leiomyoma cells. No significant effect of IL6 was observed in myometrium cells. Collagen-1 protein was also affected significantly in the leiomyoma cells on use of JAK/STAT-3 modulators. An increase of 2.21+/-0.052 fold was observed on direct activation of the pathway in leiomyoma cells. Increase were also observed in ECM protein fibronectin in response to the JAK/STAT-3 modulators. Response of transforming growth factor beta3 (TGFβ3) protein, involved in fibrotic specific pathway, on use of JAK/STAT-3 modulators indicated that both pathways may interact to regulate the production of the ECM proteins in leiomyomas. Direct effect of inflammatory cytokine IL6, on leiomyoma and not myometrium cells indicate that this differential response to IL6 and JAK/STAT3 pathway may be the key to increased ECM production and growth in uterine leiomyomas.

Simvastatin inhibits RhoA activation, collagen expression and gel contraction in human leiomyoma cells

Simvastatin inhibits RhoA activation, collagen expression and gel contraction in human leiomyoma cells

Mifepristone inhibits IGF-1 signaling pathway in the treatment of uterine leiomyomas.

PurposeTo investigate the role of IGF-1 signaling pathway in the treatment of uterine leiomyomas with mifepristone.Patients and methodsFrom October 2015 to December 2018, 50 patients with uterine leiomyoma were included in this study. Overexpression or siRNA of IGF-1 in primary human uterine leiomyoma cells were treated with or without mifepristone. MTT was used to evaluate cell viability in assays of cell proliferation and cytotoxicity. IGF-1 expression in the cells was measured with real-time RT-PCR and Western blotting and manipulated with lentivirus ectopic overexpression or siRNA silencing.ResultsInhibition of cell viability by mifepristone was found dependent on drug concentration and treatment time. IGF-1 and phosphorylation-ERK1/2 expression were decreased, while phosphorylation-AKT expression was increased after mifepristone treatment. IGF-1 significantly promoted cell growth, while IGF-1 knockdown and mifepristone showed synergistic inhibition effects on cell growth. The overexpression of IGF-1 reversed the inhibition of cell growth and ERK1/2 phosphorylation but showed no effect on AKT phosphorylation.ConclusionOur study for the first time demonstrated that IGF-1 signaling via ERK1/2 appears to be an important target of mifepristone in the treatment of uterine leiomyomas, which may provide a new approach to avoid leiomyoma re-growth after cessation of mifepristone.

Effect of simvastatin on integrin-β1 and its downstream mediators in human leiomyoma cells

Effect of simvastatin on integrin-β1 and its downstream mediators in human leiomyoma cells

A nongenomic mechanism for "metalloestrogenic" effects of cadmium in human uterine leiomyoma cells through G protein-coupled estrogen receptor.

Cadmium (Cd) is a ubiquitous environmental metal that is reported to be a "metalloestrogen." Uterine leiomyomas (fibroids) are estrogen-responsive gynecologic neoplasms that can be the target of xenoestrogens. Previous epidemiology studies have suggested Cd may be associated with fibroids. We have shown that Cd can stimulate proliferation of human uterine leiomyoma (ht-UtLM) cells, but not through classical estrogen receptor (ER) binding. Whether nongenomic ER pathways are involved in Cd-induced proliferation is unknown. In the present study, by evaluating G protein-coupled estrogen receptor (GPER), ERα36, and phospho-epidermal growth factor receptor (EGFR) expression in human tissues, we found that GPER, ERα36 and phospho-EGFR wereallhighly expressed in fibroids compared to patient-matched myometrial tissues. In ht-UtLM cells, cell proliferation was increased by low doses of Cd (0.1µM and 10µM), and this effect could be inhibited by GPER-specific antagonist (G15) pretreatment, or silencing (si) GPER, but not by siERα36. Cd-activated MAPK was dependent on GPER/EGFR transactivation, through significantly increased phospho-Src, matrix metalloproteinase-2 (MMP2) and MMP9, and heparin-binding EGF-like growth factor (HB-EGF) expression/activation. Also, phospho-Src could interact directly to phosphorylate EGFR. Overall, Cd-induced proliferation of human fibroid cells was through a nongenomic GPER/p-src/EGFR/MAPK signaling pathway that did not directly involve ERα36. This suggests that Cd may be a risk factor for uterine fibroids through cross talk between hormone and growth factor receptorpathways.

PLIN2 Functions As a Novel Link Between Progesterone Signaling and Metabolism in Uterine Leiomyoma Cells.

Uterine leiomyoma (fibroids) are the most common tumors in women. Recently, perilipin-2 (PLIN2) was identified as a critical target gene of the progesterone receptor; however, its function in the pathogenesis of fibroids is unknown. To determine the function of PLIN2 in leiomyoma cells. Tissue and primary cells from leiomyoma and myometrium were analyzed. PLIN2 function in leiomyoma was assessed using small interfering RNA. RNA-sequencing was performed to identify genome-wide effects of PLIN2 depletion. Metabolic activity was measured using the Seahorse XF96 analyzer. Real-time quantitative PCR and immunoblotting were also performed. Laboratory. Forty-one premenopausal women undergoing surgery for fibroids. Gene expression, oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and cell proliferation. PLIN2 gene expression was 2.4-fold lower in leiomyoma compared with adjacent myometrium, suggesting a link between PLIN2 deficiency and fibroids. A total of 3877 genes were differentially expressed after PLIN2 knockdown. Gene ontology analysis identified metabolism as the second-highest biological process affected by PLIN2 depletion. OCR (mitochondrial respiration) and ECAR (glycolysis) were significantly upregulated after PLIN2 knockdown; PLIN2-depleted cells had a greater basal metabolic activity and higher metabolic stress response. Cell proliferation was also significantly increased after PLIN2 knockdown. PLIN2 depletion increases mitochondrial respiration and glycolysis, suggesting that PLIN2 is a critical regulator of metabolic function in leiomyoma cells. PLIN2 deficiency also reprograms leiomyoma cells to a proproliferative phenotype. These findings introduce metabolomics as an area to explore to better understand leiomyoma tumorigenesis.

Spontaneous Age-Related Histopathological Changes in Microminipigs.

Microminipigs have become an attractive animal model for toxicology and pharmacology studies and for human disease models, owing to their manageable size. Although there are numerous reports of spontaneous age-related lesions in mice, rats, dogs, and monkeys, those in minipigs are scarce. In the present study, spontaneous age-related histopathological changes were investigated using 37 microminipigs (20 males and 17 females) that were 6 months to 10 years of age. Abnormal deposits of materials were evident in several animals from 6 years of age, and these deposits included amyloid in the renal medulla, thyroid gland, and adrenal gland, hyaline droplets in glomeruli, and fibrillar inclusions in neurons. Arterial sclerosing changes (intimal thickening, intimal proliferation, and medial mineralization) and proliferative lesions (hyperplasia of hepatocytes, follicular cells, Leydig cells, and uterine endometrial glands) were present at 4 years of age and beyond. Renal adenoma, uterine leiomyoma, and Leydig cell tumor were observed in several microminipigs. Moreover, glomerulosclerosis, renal interstitial fibrosis, thymic involution, and adrenocortical cell vacuolation were common in aging microminipigs. Since knowledge of age-related changes is helpful for pathologists, the basic information obtained in this study will be a useful reference for all future toxicity evaluations in microminipigs.

Progesterone receptor antagonist provides palliative effects for uterine leiomyoma through a Bcl-2/Beclin1-dependent mechanism.

Uterine leiomyoma is the most common benign smooth muscle tumor of uterus in women of reproductive age, with a high lifetime incidence. Nowadays, the exploration on the pharmacotherapies, such as progesterone receptor antagonist (PRA) requires more attention. Hence, the current study aimed to examine whether mifepristone, a PRA, influences the autophagy and apoptosis of uterine leiomyoma cells. Primary uterine leiomyoma cells were collected from 36 patients diagnosed with uterine leiomyoma to establish PR-M-positive (PR-M[+]) cells. The lentiviral vector overexpressing or silencing PR-M was subsequently delivered into one part of PR-M(+) cells in order to evaluate the role of PR-M in PR-M(+) cells. The results obtained revealed that cell viability was increased, while cell autophagy and apoptosis were diminished in the PR-M(+) cells treated with overexpressed PR-M, whereby the Bcl-2 level was elevated and the level of Beclin1 was reduced. An opposite trends were identified following treatment with knockdown of PR-M. Mifepristone at different concentrations (low, moderate, or high) was then applied to treat another part of the PR-M(+) cells. Mifepristone was identified to promote cell autophagy and apoptosis, decrease Bcl-2 level and increase Beclin1 level, accompanied by weakened interaction between Bcl-2 and Beclin1. Moreover, these effects of mifepristone on PR-M(+) cells were enhanced with increasing of the concentration. Taken together, the present study present evidence indicates the ability of PRA to regulate the Bcl-2/Beclin1 axis, ultimately promoting the autophagy and apoptosis of uterine leiomyoma cells, highlighting that PRA serves as a promising therapeutic target for the treatment of uterine leiomyoma.

Abstract 3022: Dissecting rapamycin resistance in a Tsc2-null rat leiomyoma cell line developed in a murine xenograft

Abstract Despite the identification of mTOR hyperactivation as the main biochemical defect downstream of TSC1/TSC2 inactivation in Lymphangioleiomyomatosis (LAM) and Tuberous Sclerosis Complex (TSC), and the approval of rapamycin and rapalogs for the treatment of the related lesions, these drugs are cytostatic and continued treatment is required for clinical benefit. The latter raises the possibility of acquired drug resistance over long-term use. To explore the mechanisms leading to rapamycin resistance in LAM/TSC, we xenografted SCID mice with ELT3 cells (Tsc2-null derived from an Eker rat uterine leiomyoma), explanted a tumor that was not responsive to rapamycin, and generated cell line ELT3-245. The cell growth of ELT3-245 is not inhibited by rapamycin in vitro. SCID mice inoculated with ELT3-245 form palpable tumors significantly faster, compared to ELT3 cells, respond very poorly to rapamycin at the initial stages of treatment, but soon become non-responsive. In addition, these cells form macroscopically visible metastases to the lungs of ELT3-245 tumor-bearing mice. We performed global gene expression profiling, comparing ELT3-245 to ELT3 cells, and found that the rapamycin-resistant cells have decreased expression of genes associated with epithelial cells and increased expression of genes associated with mesenchymal-like characteristics. Relative gene expression of Myc, Egfr, Akt2, Jun, and Ocln were assessed by RT-qPCR in ELT3 vs ELT3-245 after 24h of treatment with 20 nM rapamycin or DMSO. Important discrepancies include a 2.8-fold increased expression of Egfr under rapamycin treatment in ELT3 cells but not in ELT3-245; however, ELT3-245 had a 15-fold higher expression of Egfr prior to treatment, compared to ELT3. In addition, ELT3-245 cells strikingly increase expression of Myc when challenged with rapamycin, compared to ELT3. Similarly, Akt2 is increased considerably under rapamycin treatment in ELT3-245 and is already higher prior to treatment compared to ELT3. Finally, Ocln expression was at least 100-fold reduced in ELT3-245, compared to ELT3, both under rapamycin treatment and no-treatment conditions. These results suggest that Myc, Egfr, Akt2, and Ocln are among the genes highly relevant to acquired rapamycin resistance in this Tsc2-null rat leiomyoma cell line and point toward the pathways that need to be interfered with in order to overcome it. Citation Format: Natalia Filippidou, Mathildi Valianou, Daniel L. Johnson, John J. Bissler, Aristotelis Astrinidis. Dissecting rapamycin resistance in a Tsc2-null rat leiomyoma cell line developed in a murine xenograft [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3022.

CANCER ASSOCIATED fiBROBLASTS (CAFS) INflUENCE TISSUE INVASION ON SALIVARY GLAND MUCOEPIDERMOID CARCINOMA (MEC) CELLS

Objectives MEC is the most common salivary gland malignancy. Although prognosis is mostly based on TNM status, histologic grade is also used as a parameter to determine treatment. CAFs have been reported to influence worse behavior in several malignancies including head and neck squamous cell carcinoma. We noticed the presence of CAF-like cells, displaying immunohistochemical positivity for alpha smooth muscle actin, in some MECs with bad outcome and we hypothesize that CAFs may influence MEC aggressiveness. Therefore, we investigated tissue invasion using the organotypic 3D human leiomyoma model and cell migration using the Incucyte® system with a gel derived from human leiomyomas (myogel). MEC cell lines HMC2 and UTMUC1, derived from high grade tumors, were cultivated alone or co-cultured with CAFs in order to evaluate if CAFs would influence MEC cells invasion and migration. Cells were cultivated on top of human leiomyoma discs for 14 days to allow invasion. Discs were fixed in 10% buffered formalin, processed and 3 micrometer tissue slices were prepared and submitted to immunohistochemical reaction with a pan-cytokeratin antibody (clone AE1/AE3). The number of invasive cells was determined by counting invasive cells under light microscope. Invasion was studied using a wound scratch assay coupled with a live camera and data obtained was analyzed using software provided by the manufacturer. Findings Both MEC cell lines (HMC2 and UTMUC1) displayed a significant increase in tissue invasion when co-cultured with CAFs compared to when they were cultured alone. Only HMC2 cell line presented a significant increase in migration when co-cultured with CAFs. Conclusion CAFs significantly increase MEC cell lines invasion and migration. The presence of CAFs deserves further investigation in MEC tumor samples and it may correlate with tumor behavior and clinical outcome.

A Culture System Similar to the Embryonal Microenvironment Supports Transdifferentiation in Human Leiomyoma Cells

Current research on stem cells and regenerative medicine indicates new perspectives on the relationship between differentiation and gene information. Induced pluripotent stem (iPS) cells need the artificial gene expression of the somatic cell, which is related to initialization. Paradoxically, that means that cell differentiation depends on almost all the gene information stored precisely in the nucleus of a somatic cell, plus the transformation of gene expression. Our research team tried to identify the culture conditions in the transdifferentiation of human leiomyoma cells, closely similar to the early embryonal stage, composed of various factors (hypoxia, non-serum, and regulation of cell adhesion molecules such as Wnt/β-catenin signaling). As a result, inhibition of Wnt/β-catenin signaling under serum starvation and hypoxia induces adipocytic transdifferentiation in human leiomyoma cells. Here we explain this unique culture system, referring to the components of intracellular mechanisms and the extracellular microenvironment in embryo development.