In this study, we compared the selected cell cycle regulators and AKT in ULM with three different driver gene mutations and found that only the tumors had high AKT activity, high cell proliferation indices (Ki-67), and significantly lower p16 expression (Figure 2). mutations are the second most common driver mutations in ULM, accounting for 10-15% of the cases6. rare cases4, 5. Global gene expression analysis indicates that different mutant genes in ULM may target different molecular pathways5 and each mutation type may theoretically determine growth behavior, but these theories have not yet been proven. Notably, a uterus with multiple ULM can show somatic mutations of different genes and each given tumor only acquires a single driver gene mutation, since the mutations are mutually exclusive4, 6, 7. Thus, ULM consist of a group of genetically heterogeneous tumors with similar histogenesis. Such genetic differences in ULM due to different driver gene mutations may determine their histological and molecular heterogeneity and further influence tumor growth rates. However, histology and molecular pathway correlation with these driver gene mutations has not been established or characterized. overexpression in ULM is caused by a translocation between 12q and 14q8 and the early studies showed that ULM with overexpression tend to be larger and grow faster than those without alterations5, 9, 10. ULM cannot be differentiated histologically from other ULM, but it is common in a variant of ULM defined as intravascular leiomyomatosis11. Unfortunately, while many oncogenic functions of in malignant tumors are characterized12, little is known about how causes and promotes ULM development and growth. mutations are the most common somatic mutations in ULM13. MED12 is essential for activating CDK8 and modulates mediator-polymerase II interactions for transcription initiation14. Growth of ULM may require and recruitment of prominent myoma-associated fibroblasts15. ULM exhibit characteristic histologic features in leiomyomas of bizarre nuclei16, 17. Further investigation of the molecular and histological difference in ULM with different driver gene mutations may UR 1102 assist in understanding biological and medical significance and aid in clinical management. Since sex steroid hormones, cell cycle and AKT signaling are prevalent pathways for ULM growth, we aimed in this study to examine these common functional pathways in ULM with different driver mutations. We collected ULM with and mutations and examined the selected markers by immunohistochemistry. The functional correlation between AKT and was further analyzed in primary cultures of ULM. 2. MATERIALS AND METHODS 2.1 Case selection Human myometrial and leiomyoma tissues were collected from premenopausal women undergoing hysterectomy at the Northwestern University. The use of human tissue specimens was approved by the Institutional Review Board for Human Research at Northwestern University. Fresh frozen and/or formalin-fixed and paraffin-embedded tumor, and myometrial tissues were used. The genotypes of the selected ULM with MED12 mutations, HMGA2 overexpression and biallelic FH inactivation have been reported in previous studies6, 16. 2.2 Primary cell culture for ULM Subjects were only included in the study if they were not taking hormonal contraceptives or gonadotropin-releasing hormone agonists/antagonists for at least 3 months. Informed consent was obtained from all the patients participating in the study. After tissue was collected, primary myometrial and leiomyoma cells were isolated and cultured. Primary cells were cultivated in Dulbeccos modified Eagles medium/nutrient Hams Mixture F-12 (DMEM-F12) 1:1 containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37C and 5% CO2 atmosphere. Primary ULM cell cultures were maintained in Smooth Muscle Growth Medium-2 (SmGM?-2 medium) (Lonza) to avoid loss of myoma cells. 2.3 Senescence-associated -galactosidase (SA–gal) staining The cells were treated with MK2206 at 5M (Merck Sharp & Dohme Corp.) Then the cells were fixed with 2% CORO1A formaldehyde plus 0.2% glutaraldehyde and were stained with -galactosidase staining solution (citric acid/sodium phosphate solution, potassium ferrocyanide, potassium ferricyanide, X-gal, pH6). They were incubated at 37C overnight in a dry incubator and the reactions were terminated when the cells were stained blue-green, as visualized under an inverted bright-field microscope. The cells were also stained with DAPI (4, 6-Diamidino-2-Phenylindole, Dihydrochloride) to show the nucleus. Three images were taken randomly under each treatment condition and then the percentage of the cells that were positive for -galactosidase was calculated. 2.4 SDS-PAGE and Western blotting Protein lysates were extracted from myometrial UR 1102 and leiomyoma cells using RIPA lysis and extraction buffer with protease and phosphatase inhibitors (Thermo Fisher Scientific). The protein concentration was determined using BCA Protein Assay kit (Thermo Fisher Scientific). Equal amounts of proteins were subjected UR 1102 to SDS-PAGE and subsequently transferred to.