Inactivation of AKT induces cellular senescence in ...

Inactivation of AKT induces cellular senescence in uterine leiomyoma Xiaofei Xu 1,3 , Zhenxiao Lu 2 , Wenan Qiang 2 , Beihua Kong 3 , J. Julie Kim 2 , Jian-Jun Wei 1 1. Department of Pathology, Northwestern University Feinberg School of Medicine; 2. Department of Obstetrics and Gynecology, Northwestern University Feinberg School of Medicine; 3. Department of Obstetrics and Gynecology, Shandong University, China Uterine leiomyomas (fibroids) are a major public health problem. Current medical treatments with GnRH analogues do not provide long term benefit. T hus, permanent shrinkage or inhibition of fibroid growth via medical means remains a challenge. T he AKT pathway is a major growth and survival pathway for fibroids. We propose that AKT inhibition results in a transient regulation of specific mechanisms that ultimately drive cells into cellular senescence or cell death. In this study, we investigated specific mechanisms of AKT inhibition that resulted in senescence. We observed that administration of MK-2206, an allosteric AKT inhibitor, increased levels of reactive oxygen species (ROS), upregulated the microRNA miR-182, and several senescence associated genes (including p16, p53, p21, and galactosidase), and drove leiomyoma cells into stress induced premature senescence (SIPS). Moreover, induction of SIPS was mediated by HMGA2 which co-localized to senescence-associated heterochromatin foci (SAHF). T his study provides a conceivable molecular mechanism of SIPS by AKT inhibition in fibroids.

Uterine leiomyomas (fibroids) are smooth-muscle tumors of the myometrium that occur in 77% of women in the United States (1, 2). It causes significant morbidity from profuse menstrual bleeding and pelvic discomfort as well as reproductive dysfunction and accounts for over 200,000 hysterectomies each year. Treatment by gonadotropin release hormone (GnRH) agonists and antagonists can reduce/shrink tumor size, but fibroids return to original size after termination of the therapy (2, 3). Furthermore, the side-effects of antihormonal treatment are not ideal for long time usage, and therefore only preoperative treatment is recommended (4). Long-term therapies aimed at permanent shrinkage or inhibition of fibroid growth are currently not available in the US. Thus, nonhormonal, noninvasive therapeutic modalities are an attractive alternative. We have previously shown that the AKT pathway is activated in approximately 30% of fibroids as determined by IHC (5). This is most likely an underestimation due to the insensitivity of IHC staining as more sensitive western blot analysis reveal that all leiomyoma tissues tested have remarkably higher phosphorylated (Ser473)-AKT levels than matched myometrial tissues, establishing AKT activation as a potential molecular signature of leiomyomas (6 – 8). While the AKT pathway is known to promote proliferation and survival in many tissues (9), the underlying molecular mechanisms are cell-specific and complex involving the interaction of numerous proteins, genes and pathways. Development of therapeutic modalities by inactivation of the AKT pathway may provide a valuable nonhormonal treatment option for this common and un- derstudied disease affecting millions of reproductive aged women in the United States and worldwide. Inhibition of mammalian target of Rapamycin (mTOR), a downstream of AKT pathway, has been reported (10). MK-2206 is an allosteric AKT inhibitor and is currently in clinical trials for some solid tumors. Preclinical, MK-2206 is effective in reducing xenograft growth in models of breast, prostate, nonsmall cell lung, ovarian cancers and more recently, uterine leiomyomas (11–13). In phase 1 trials, this inhib-

itor was shown to have antitumor properties, while causing minor side effects, including skin rash, nausea, pruritus, hyperglycemia and diarrhea (14). Currently, phase I and II trials are being conducted with MK-2206 in solid tumors and blood cancers (www.clinicaltrials.gov). The AKT pathway is tightly regulated in a normal cell depending on whether the cells undergo proliferation vs cellular senescence (15). Recent studies revealed that AKT inhibition results in stress-induced premature senescence (SIPS) in some cell types (16, 17). Naturally occurring cellular senescence is common in fibroids (18). Further- more, since the AKT pathway is commonly activated in fibroids (6 – 8) and it is key for survival of leiomyomas (11), we hypothesized that inhibition of AKT can lead to cellular senescence in leiomyoma. In this study, we investigated the mechanisms by which AKT inhibition induces senescence in fibroid cells in vitro. We observed that administration of MK-2206, an AKT inhibitor, increased levels of reactive oxygen species (ROS), upregulated miR-182, HMGA2 and several senescence associated genes, and promoted SIPS in leiomyoma cells.

Materials and Methods Tissue Collection and Cell Culture Leiomyoma tissues were collected from premenopausal women undergoing hysterectomy or myomectomy at North- western University Prentice Women’s Hospital, Chicago IL according to an IRB approved protocol. Women included in the study were not taking hormonal contraceptives or gonadotropin releasing hormone antagonist or agonist (GnRHa) at least 3 months prior to tissue collection. Consent was granted by all women included in the study. Tumor sections adjacent to the peripheral zone were collected (19) and minced into small pieces and digested with collagenase A and DNAase (Sigma Aldrich) for 6 hours on a 37°C tissue shaker. The digested material was fil- tered to obtain single-cell suspension. The primary cells were cultured in smooth muscle cell basal medium (SmBM ; Clonetics, Lonza Group, Switzerland) no longer than 10 days. All experi- ments involving primary leiomyoma cells were repeated with cells from at least 3 different patient tissues. M ore than 10 leiomyoma tissues were collected for this study. The human myometrial cell line myo-hTert and the leiomyoma cell line DD-HLM cells were kindly provided by C. M endelson (UT Southwestern) and A. Al Hendy (M eharry), respectively. Cells were maintained in advanced DM EM /F12 (1:1) (Invitrogen) medium supplemented with 10% fetal bovine serum (USA Scientific). Chem) or doxorubicin (Sigma). Cells were also pulse-treated once each hour for 5 hours with hydrogen peroxide (H 2 O 2 ; 100 M ) and cultured for up to 48 hours. In some cases, N-Acetyl- Cysteine (NAC; Sigma) were added to the cells 30 minutes prior to cotreatment with M K-2206 for an additional 1h.

Three dimensional leiomyoma cell cultures Primary cells or immortalized cells were trypsinized from the plate and washed twice with phosphate-buffered saline (PBS)/ solution (PBS). Cell count and viability were confirmed using 0.4% Trypan Blue solution. Cells were suspended into rat type IV collagen (1 mg/mL, BD Biosciences, San Jose, CA) at 10 6 per 40 L. The 40 L cell-collagen mixture was dropped onto the M illicell Hanging Cell Culture Insert (pore size 3.0 m, M illipore) placed in 24-well plate, and incubated at 37°C in a humidified atmosphere of 95% air and 5% CO 2 . After half an hour, cultured medium was added into both the insert and the well and the system was incubated overnight and then treated with M K- 2206, BEZ, DOX or H 2 O 2 . Collagenenclosed cells were sec- tioned consecutively (5 m) on a freezing microtome and stored at –20°C for future use.

siRNA transfection The siRNAs for HMGA2 were purchased from Invitrogen Life Technologies (Carlsbad, CA), and have been tested in a previous study (20). Briefly, cells were placed in 6-well plate (2 10 5 per well) in media without antibiotics for 24 hours. After 70% confluence, cells were transfected with HMGA2-siRNA (60 pmol/well) or control small RNA (Block-iT fluorescent dou- ble-stranded random 22mer RNA from Invitrogen), using Lipofectamine 2000 according to the manufacturer’s protocol. After transfection, cells were harvested and analyzed at the indicated times.

ROS (reactive oxygen species) assays Intracellular ROS was detected using the O2- -sensitive fluorescent probe dye dihydroethidium ((DHE; Invitrogen). Briefly, cells (5 x 10 4 ) were seeded onto 6-well plate one day prior to detection. Cells were treated with NAC with or without M K2206. 10uM of DHE were then added for 20min. Cells were washed in HBSS and the intracellular ROS levels represented by the percentage of cells with DHE staining were visualized under a Zeiss Axiovert fluorescent microscope.

Senescence associated stain Cells were seeded onto coverslips placed in 6-well plate overnight and then treated with the test compounds: M K2206 (2 M ), H 2 O 2 (100 M ), or doxorubicin (DOX, 0.2 g/mL). Cells were fixed with 2% formaldehyde 0.2% glutaraldehyde in PBS at room temperature for 3–5 minutes. After washing in PBS, cells were stained with staining solution containing 1 mg/mL X-gal and incubated in a CO 2 -free incubator at 37°C for 16 hours. Blue cells were counted under microscope and statistically analyzed. Three randomly selected fields (1 mm 2 ) of images were captured to count the senescence rate (%).

2

normal goat serum for 30 minutes and then incubated with specific primary antibodies including mouse antihuman phospho- H2AX (1:200; M illipore) or rabbit antihuman HM GA2 (1:100, BioCheck) at 37°C for 1 hour. M ouse or rabbit IgG was used as the negative control. After washing in PBS, cells were incubated with TRITC-conjugated goat antimouse or goat antirabbit secondary antibody at room temperature for 1 hour. The nuclei were counterstained with 4 ,6-diamidino-2-phenylindole (DAPI) to visualize the senescence-associated heterochromatin foci (SAHF) (21). Three randomly selected fields of fluorescent images were captured under a fluorescence microscope and a total of 50 cells were counted in each sample to calculate the percentage of positive-staining cells. The percentage of phospho- H2AX-positive cells were calculated to demonstrate the level of DNA damage caused by different stimulation, and the percentage of SAHF-positive cells were calculated to indicate the senescent cells.

RNA extraction and quantitative real-time RT-PCR Total RNA was extracted using the TRIzol (Invitrogen) or miRNA extraction kit (Ambion, TX) according to the manufacturer’s instructions. 1 g of total RNA or 50 ng small RNA were reverse-transcribed to cDNA in a 20 L volume using an Ad- vantage RT for PCR Kit (Clontech, M ountain View, CA) or miRNA kit (Ambion). -actin or U6 were used as internal con- trols for all PCR. Quantitative real-time PCR was performed with SYBR Green real-time PCR master mix (Bio-Rad, Hercules, CA) using a M yiQ and iQ5 real-time PCR Detection System with sequence specific primers. All PCR were run for 40 cycles (95°Cfor 15 seconds, 60°Cfor 1 minute) after a10-minute incu- bation at 95°C. The fold change in expression of each gene was calculated with the change in cycle threshold value method ( Ct). The primers for tested genes are summarized in S Table 1.

Western blotting Cultured cells were harvested and lysed (ie, in mammalian protein extraction reagent (Thermo Scientific, Rockford, IL) supplemented with protease and phosphatase inhibitors (Sigma, St. Louis, M O) on ice. Total proteins (30 g) were separated by SDS-PAGE and electrotransferred onto polyvinylidene fluoride membrane. The membrane was incubated with primary antibodies overnight at 4°C (Suppl Table 2). Proteins of interest were detected with the appropriate horseradish peroxidase-conjugated secondary antibodies and developed using the ECL PLUS kit (Amersham Biosciences, Piscataway, NJ).

Statistical analysis Continuous data were measured for means and standard er- rors in triplicate experimental samples. The data including 3 or more groups were checked for the normality and then preceded to one-way ANOVA analysis. Student’s t test was used for com- parisons between 2 groups. Significance for noncontinuous data was calculated by X 2 analysis. P .05 was considered significant.

Results Inhibition of AKT results in increased cellular leiomyoma cells are largely unknown. In our previous studies, we found that activation of AKT was essential for leiomyoma growth and survival (11). Evidence is emerging that AKT can protect against stress induced premature senescence (SIPS) (16). We proposed that inhibiting the AKT pathway triggers multiple steps of stress responses that ultimately navigate cells into cellular senescence. Primary leiomyoma cells (n 3) were treated with in- creasing concentrations of MK-2206 for 48 hours and protein levels of p(Ser473)-AKT and its downstream effector pPARS40 were measured by Western blot (Figure 1A). We found that with as little as 0.01uM MK-2206 treatment, the levels of p(Ser473)-AKT and its effector pPARS40 was decreased. 1uM and high dose of MK-2206 could completely inhibit pAKT and pPARS40 (Figure 1A). To investigate whether inhibition of AKT induced senescence, leiomyoma cells were treated with MK-2206 and subjected to -galactosidase staining, a widely used biomarker for senescent cells. Upon treatment with MK- 2206, -galactosidase stained cells were evident in both DD-HLM (Figure 1B) and primary (Figure 1C) leiomyoma cells. The percentage of -galactosidase stained DD- HLM and primary leiomyoma cells with MK-2206 treatment were significantly higher than untreated cells. Doxorubicin treatment was added as a positive control for inducing senescence. Similarly, the percentage of -galactosidase stained cells increased for the myometrial cell line, myo-hTert (S Figure 1B). Another inhibitor of the AKT pathway, GDC-0980 which is specifically a dual PI3K and mTOR inhibitor, (1 M) increased -galactosidase staining in DD-HLM cells (S Figure 1A), demonstrating that induction of senescence was not specific to MK-2206. Next, we examined and counted the percentage of senescence associated heterochromatin foci (SAHF) which are characteristic structural changes that occur in primary leiomyoma cells undergoing senescence (22). They appear as punctate, focal staining within the nuclei stained with DAPI. Approximately 32% of cells exhibited SAHF when treated with MK-2206 compared to 8% in control cells (Fig, 1D). Doxorubicin (DOX) was used as a positive control for inducing SIPS. Finally, we examined the expression of genes associated with senescence using real-time RT-PCR. When leiomyoma cells were treated with MK-2206, the senescence associated genes, P16, P21 and P53 were significantly upregulated (Figure 1E). These data demonstrate that inhibition of AKT in leiomyoma cells induces senescence which resembles SIPS.

3

4

dria (11). Since mitochondrial damage can increase levels of ROS and ROS is also a well-known stress factor for DNA and mitochondrial damage for promoting SIPS, leiomyoma cells were treated with MK-2206, and intracellular ROS production was measured by DHE. In response to MK-2206 as well as the dual PI3K/mTOR inhibitor, BEZ- 235, ROS levels increased in primary leiomyoma cells (Figure 2A).To demonstrate that ROS can directly induce SIPS, leiomyoma cells were treated with 100 M H 2 O 2 and as a result, more than 50% of cells stained positively for -galactosidase indicating SIPS (Figure 2B). Quanti- tation of -galactosidase positive cells showed increased percentage of cells staining blue with H 2 O 2 at 8 hour and 24 hour. H 2 O 2 also increased senescence associated genes, GLB1, P16, and P53 (Figure 2C). Treatment of cells with the antioxidant, N-Acetyl-Cysteine (NAC) attenuated MK-2206 induced P16 expression, but not P53 expression (Figure 2D). MK-2206-mediated ROS production regulates microRNAs expression MicroRNAs play a key role in response to stress factors. Among them, miR-182 and miR-200s are critical mole- cules in stress-mediated cellular function and they are con- sistently upregulated by ROS (23, 24). MiR-182 plays a major role in DNA damage response (DDR) through reg- ulating several DDR related gene expression, including BRCA1, FOXO3a and HMGA2 (25, 26). MiR-200s reg- ulate cellular senescence and cell proliferation (24, 27). The regulation of these two microRNAs by AKT, specifically, when AKT is inactivated, has never been studied. First, we examined miR-182 and miR200a/c expression when DD-HLM cells were treated with H 2 O 2 . Consistent with results found in other cell types, miR-182 and miR200a/c were induced in DD-HLM cells with H 2 O 2 treatment (Figure 3A). Similarly, miR-182 and miR-200a/c expression increased in leiomyoma cells treated with MK- 2206 in a dose-dependent manner (Figure 3B). The

5

addition of NAC to MK-2206 treated cells blunted the expression of the three miRNAs suggesting that ROS is mediating this increase (Figure 3A). In addition to miR- 182 and miR200a/c, other stress related miRNAs were examined. MiR-182 and its family members, miR-183 and miR-96 were increased in response to MK-2206. Interestingly, miR-29b was significantly downregulated in leiomyoma

cells treated with H 2 O 2 (S Figure 2). MK-2206 induced SIPS upregulates HMGA2 which localizes to SAHF HMGA2 is an oncogene and is overexpressed in a small portion of leiomyomas due to t (12, 14) translocation (28). In leiomyomas, HMGA2 is thought to promote cell proliferation (28) and prevents senescence through inhibition of genes associated with senescence (29). However, it has been shown in other cells that undergo oncogene stress that HMGA2 is upregulated and coordinates with other senescence associated factors such as P16 for cellular senescence (22). To investigate the role of HMGA2 in SIPS induced by AKT inhibition, HMGA2 expression was examined in leiomyoma cells treated with MK-2206. As shown in Figure 4A, HMGA2 expression was significantly increased in a dosedependent manner when AKT was inactivated by MK-2206. Consistent with MK-2206, H 2 O 2 treatment also increased HMGA2 expression (Figure 4B). Cotreatment with NAC and MK-2206 slightly reduced HMGA2 expression compared to MK-2206 (Fig- ure 4C). These findings suggest that ROS may be involved but not the major mediator of MK-2206 upregulation of HMGA2. To determine the localization of HMGA2 in senescent cells, immunofluorescent staining for HMGA2 was performed in leiomyoma cells treated with MK-2206. In the untreated cells, HMGA2 levels were low and found evenly distributed throughout the entire nuclei (Figure 4D). In contrast, leiomyoma cells treated with MK-2206 showed punctate localized staining of DAPI in the nuclei also termed as senescence associated heterochromatin foci (SAHF). Furthermore, there was higher immunoreactivity for HMGA2 mainly localized in these punctate foci (Fig- ure 4D). We therefore proposed that HMGA2 upregulation and localization to SAHF was required for MK-2206 induced SIPS in leiomyoma when AKT is inactivated. In order to determine whether HMGA2 plays a functional role in SIPS, HMGA2 was knocked down using siRNA, and cells were treated with MK-2206 or H 2 O 2 . HMGA2 knock down was efficient even in response to MK-2206, H 2 O 2 or DOX which upregulates its levels (Figure 5C). In response to HMGA2 knockdown, the SAHF bodies, as visualized

6

evident (Figure 5A). Quantitation of the percentage of heterochromatin positive cells revealed a significant de-

7

crease when HMGA2 was silenced (Figure 5B) (P .05). These observations strongly suggest that HMGA2 plays an active role in promoting SAHF formation and ultimately promotes senescence in response to AKT inhibition.

Discussion Leiomyomas require prosurvival mechanisms to adapt to an unfavorable hypoxic microenvironment (30) and emerging evidence points to AKT as a major survival pathway for leiomyomas (9, 11). Not only is the AKT pathway activated in most leiomyomas, we demonstrated that inhibition of this pathway leads to decreased growth of leiomyoma tumors and promotes cell death (11). Inhibiting the AKT pathway results in a dynamic and transient regulation of specific pathways that ultimately navigate cells into various downstream fates, including decreased proliferation and cellular senescence. It has been shown in other cell types, that AKT can protect against senescence that is induced by RAS (16, 17). We have demonstrated in this study that inhibition of the AKT pathway in leiomyoma cells induces senescence that is reminiscent of SIPS. Several key senescence-associated genes (P16, P21 and P53) were upregulated when treated with AKT inhibitor MK-2206. P16 was induced with a very low dose of MK- 2206, while P21 and P53 upregulation required higher doses of MK-2206 (Figure 1E). We further demonstrated that AKT inhibition results in multiple cellular and molecular alterations, including increased ROS production, which induces redox sensitive microRNA expression, senescence associated gene expression and increase of SAHF, a hallmark of SIPS. Senescence is permanent growth arrest that occurs in response to either aging, called, aging replication senescence (ARS), or to stress, termed stress induced premature senescence (SIPS). The underlying causes for natural occurring SIPS in leiomyomas are largely unknown. One study proposed that cellular senescence in leiomyoma occurs in order to balance cell proliferation and stress, which are mediated by cell cycle regulators, including HMGA2 and p53(31). Induction of SIPS with AKT inhibitors may uncover a potential therapeutic modality in leiomyoma. Global microRNA profiling analyses reveal that miR- 182 is one of a few microRNAs that are induced in cells responding to stress (32) and, in particular, for ROS induced senescence in fibroblasts and other cell types (23, 24). Here, we demonstrated that miR-182 and miRsponsible for the senescent phenotype. Based on our previous study showing that miR-182 upregulates expression of HMGA2 in ovarian cancer cells (25), increased miR- 182 expression in leiomyoma cells may be a major regu- lator of HMGA2 expression. Our data showed that HMGA2 expression increases in response to MK-2206 as well as H 2 O 2 . The increased expression of HMGA2 during senescence seems paradoxical as HMGA2 is a known oncogene and promotes fibroid growth (28). About 7.5%-10% of leiomyomas overex- press HMGA2 due to nonrandom chromosomal translocation (5, 6, 33–35) and this overexpression of HMGA2 has been implicated in leiomyoma tumor formation and tumor growth (28, 31, 36). In most leiomyomas without the chromosomal translocation, HMGA2 is not expressed unless the endogenous gene is induced (37). It has been shown that activity of the AKT pathway determines the fate of HMGA2 for cell proliferation or senescence in some cell types (38). This regulation mechanism seems to be true in leiomyoma, as AKT inhibition induces HMGA2 upregulation. Furthermore, HMGA2 colocalizes to the nucleus into SAHF bodies (22), which may attenuate oncogenic and mitogenic functions of HMGA2. These foci appear as compacted DNA and feature protein modifica- tions typical of transcriptionally inactive heterochromatin. Silencing HMGA2 prohibits cells from forming these foci structures and further supports the role of HMGA2 in SIPS. Yu et al (38) showed that p16 INK4A is negatively regulated by the AKT pathway in that activated AKT sup- pressed p16 INK4A expression. In this study, we noted that P16 INK4A was upregulated in primary and immortalized fibroid cells when treated with either MK-2206 or H 2 O 2 . Since senescent cells require P16 INK4A for HMGA2 mediated SAHF formation (22), increases of HMGA2 and P16 INK4A by AKT inhibition may be necessary for SIPS. In summary, we observed multiple cellular and molecular changes induced by AKT inhibition which could be involved in driving leiomyoma cells into SIPS. Our work- ing model depicted in Figure 6, is that in leiomyomas, inhibition of the AKT pathway, causes increased production of ROS and miR-182 expression, which thereby increases HMGA2 expression, which then localizes to the heterochromatin foci, and forms a complex that involves p16 INK4A . This complex prevents HMGA2 from acting as an oncogene but rather, promotes senescence, as well as structural changes that involve the formation of heterochromatin foci (SAHF). Our findings significantly further our understanding of the mechanisms of leiomyoma sur-

8

We thank Dr. Bushra Ayub for her technical assistance. This work is supported by start-up funds (JJW) and NIH P01HD057877 (JJK). Conflict of interest: Authors have nothing to disclose. Address all correspondence to: J. Julie Kim, Ph.D., Department of Obstetrics and Gynecology, Feinberg School of M edicine, Northwestern Uni- versity, 303 East Superior Street, Lurie 4 –117, Chicago, IL 60611, Phone: 312–503-5377, Fax: 312–503-0095, E-mail:; Jian-Jun Wei, M .D., Department of Pathology, Feinberg School of M edicine, Northwestern University, 251 East Huron Street, Feinberg 7–334, Chicago, IL 60611, Phone: 312–926-1815, Fax: 312–926-3127, [email protected]. Conflict of interest: Authors have nothing to disclose. Grants: This work is supported by start-up funds (JJW) and NIH P01HD057877 (JJK).

References 1. Baird DD, Dunson DB, Hill MC, Cousins D, Sche ctman JM. High cumulative incidence of uterine leiomyoma in black and white women: ultrasound evidence. Am J Obstet Gynecol. 2003;188:100 –107. 2. Bulun SE. Uterine fibroids. N Engl J Med. 2013;369:1344 –1355. 3. Frie dman AJ, Daly M, June au-Norcross M, Gle ason R, Re in MS, Le Boff M. Long-term medical therapy for leiomyomata uteri: a pro- spective, randomized study of leuprolide acetate depot plus either oestrogen-progestin or progestin ‘add-back’ for 2 years. Hum Re- prod. 1994;9:1618 –1625. 4. 2008 ACOG practice bulletin. Alternatives to hysterectomy in the management of leiomyomas. Obstet Gynecol 112:387– 400. 5. Pe ng L, We n Y, Han Y, We i A, Shi G, Miz uguchi M, Le e P, He rnando E, Mittal K, We i JJ. Expression of insulin-like growth factors (IGFs) and IGF signaling: molecular complexity in uterine leiomyomas. Fertil Steril. 2009;91:2664 –2675. 6. Kovacs KA, Le ngye l F, Kornye i JL, Ve rte s Z, Sz abo I, Sume gi B, Ve rte s M. Differential expression of Akt/protein kinase B, Bcl-2 and Bax proteins in human leiomyoma and myometrium. J Steroid Biochem Mol Biol. 2003;87:233–240. 7. Pe ng L, We n Y, Han Y, We i A, Shi G, Miz uguchi M, Le e P, He rnando E, Mittal K, e t al. Expression of insulin-like growth factors (IGFs) and IGF signaling: molecular complexity in uterine leiomyomas. Fertility and Sterility. 2009;91:2664 –2675. 8. Varghe se BV, Koohe stani F, McWilliams M, Colvin A, Gune war- de na S, Kinse y WH, Nowak RA, Nothnick WB, Che nnathukuz hi VM. Loss of the repressor REST in uterine fibroids promotes aber- rant G protein-coupled receptor 10 expression and activates mammalian target of rapamycin pathway. Proc Natl Acad Sci U S A. 2013;110:2187–2192. 9. Datta SR, Brune t A, Gre e nbe rg ME. Cellular survival: a play in three Akts. Genes, development. 1999;13:2905–2927. 10. Crabtre e JS, Je linsky SA, Harris HA, Choe SE, Cotre au MM, Kim- be rland ML, Wilson E, Saraf KA, Liu W, McCampbe ll AS, Dave B, Broaddus RR, Brown EL, Kao W, Skotnicki JS, Abou-Gharbia M, Winne ke r RC, Walke r CL. Comparison of human and rat uterine leiomyomata: identification of a dysregulated mammalian target of rapamycin pathway. Cancer Res. 2009;69:6171– 6178. 11. Se fton EC, Q iang W, Se rna V, Kurita T, We i JJ, Chakravarti D, Kim JJ. 2013 MK-2206, an AKT Inhibitor, Promotes Caspase-Indepen- dent Cell Death and Inhibits Leiomyoma Growth. Endocrinology 12. Me ng J, Dai B, Fang B, Be ke le BN, Bornmann WG, Sun D, Pe ng Z, He rbst RS, Papadimitrakopoulou V, Minna JD, Pe yton M, Roth JA. Combination treatment with MEK and AKT inhibitors is more effective than each drug alone in human non-small cell lung cancer in vitro and in vivo. PloS one. 2010;5:e14124. 13. Hirai H, Sootome H, Nakatsuru Y, Miyama K, Taguchi S, Tsujioka K, Ue no Y, Hatch H, Majumde r PK, Pan BS, Kotani H. MK-2206, an allosteric Akt inhibitor, enhances antitumor efficacy by standard chemotherapeutic agents or molecular targeted drugs in vitro and in vivo. Molecular cancer therapeutics. 2010;9:1956 –1967. 14. Yap TA, Yan L, Patnaik A, Fe are n I, O lmos D, Papadopoulos K, Baird RD, De lgado L, Taylor A, Lupinacci L, Riisnae s R, Pope LL, He aton SP, Thomas G, Garre tt MD, Sullivan DM, de Bono JS, Tolche r AW. First-in-man clinical trial of the oral pan-AKT inhibitor MK-2206 in patients with advanced solid tumors. J Clin Oncol. 2011;29:4688 – 4695. 15. Bononi A, Agnole tto C, De Marchi E, Marchi S, Pate rgnani S, Bo- nora M, Giorgi C, Missiroli S, Pole tti F, Rime ssi A, Pinton P. Protein kinases and phosphatases in the control of cell fate. Enzyme re- search. 2011;2011:329098. 16. Ke nne dy AL, Morton JP, Manoharan I, Ne lson DM, Jamie son NB, Pawlikowski JS, McBryan T, Doyle B, McKay C, O ie n KA, Ende rs GH, Zhang R, Sansom O J, Adams PD. Activation of the PIK3CA/ AKT pathway suppresses senescence induced by an activated RAS oncogene to promote tumorigenesis. Molecular cell. 2011;42:36 – 49. 17. Brune t A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Ande rson MJ, Arde n KC, Ble nis J, Gre e nbe rg ME. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell. 1999;96:857– 868. 18. Lase r J, Le e P, We i JJ. Cellular senescence in usual type uterine leiomyoma. Fertil Steril. 2010;93:2020 –2026. 19. We i JJ, Zhang XM, Chiriboga L, Ye e H, Pe rle MA, Mittal K. Spatial differences in biologic activity of large uterine leiomyomata. Fertil Steril. 2006;85:179 –187. 20. Wu J, Liu Z, Shao C, Gong Y, He rnando E, Le e P, Narita M, Mulle r W, Liu J, We i JJ. HMGA2 overexpression-induced ovarian surface

9

TJ, Zhang R. Oncogenic Ras Regulates BRIP1 Expression to Induce Dissociation of BRCA1 from Chromatin, Inhibit DNA Repair, and Promote Senescence. Dev Cell. 2011;21:1077–1091. 22. Narita M, Narita M, Kriz hanovsky V, Nune z S, Chicas A, He arn SA, Mye rs MP, Lowe SW. A novel role for high-mobility group a proteins in cellular senescence and heterochromatin formation. Cell. 2006;126:503–514. 23. Li G, Luna C, Q iu J, Epste in DL, Gonz ale z P. Alterations in mi- croRNA expression in stress-induced cellular senescence. Mech Age- ing Dev. 2009;130:731–741. 24. Mage nta A, Ce ncioni C, Fasanaro P, Zaccagnini G, Gre co S, Sarra- Fe rraris G, Antonini A, Marte lli F, Capogrossi MC. miR-200c is upregulated by oxidative stress and induces endothelial cell apopto- sis and senescence via ZEB1 inhibition. Cell death and differentia- tion. 2011;18:1628 –1639. 25. Liu Z, Liu J, Se gura MF, Shao C, Le e P, Gong Y, He rnando E, We i JJ. MiR-182 overexpression in tumourigenesis of high-grade serous ovarian carcinoma. J Pathol. 2012;228:204 –215. 26. Moskwa P, Buffa FM, Pan Y, Panchakshari R, Gottipati P, Musche l RJ, Be e ch J, Kulshre stha R, Abde lmohse n K, We instock DM, Gorospe M, Harris AL, He lle day T, Chowdhury D. miR-182-mediated downregulation of BRCA1 impacts DNA repair and sensi- tivity to PARP inhibitors. Mol Cell. 2011;41:210 –220. 27. Zavadil J, Ye H, Liu Z, Wu J, Le e P, He rnando E, Sote ropoulos P, Torune r GA, We i J-J. 2010 Profiling and functional analyses of microRNAs and their target gene products in human uterine leiomyomas. PloS one 5. 28. Hodge JC, Kim TM, Dre yfuss JM, Somasundaram P, Christacos NC, Rousse lle M, Q uade BJ, Park PJ, Ste wart EA, Morton CC. Expression profiling of uterine leiomyomata cytogenetic subgroups reveals distinct signatures in matched myometrium: transcriptional profilingof the t(12;14) and evidence in support of predisposing genetic heterogeneity. Hum Mol Genet. 2012;21:2312–2329. 29. Markowski DN, He lmke BM, Be lge G, Nimz yk R, Bartnitz ke S, De iche rt U, Bulle rdie k J. HMGA2 and p14Arf: major roles in cellular senescence of fibroids and therapeutic implications. Anticancer Res. 2011;31:753–761. 30. Maye r A, Hocke l M, Wre e A, Le o C, Horn LC, Vaupe l P. Lack of hypoxic response in uterine leiomyomas despite severe tissue hyp- oxia. Cancer Res. 2008;68:4719 – 4726. 31. Markowski DN, von Ahse n I, Ne z had MH, Wosniok W, He lmke BM, Bulle rdie k J. HMGA2 and the p19Arf-T P53-CDKN1A axis: a delicate balance in the growth of uterine leiomyomas. Genes Chro- mosomes Cancer. 2010;49:661– 668. 32. Suz uki HI, Yamagata K, Sugimoto K, Iwamoto T, Kato S, Miyaz ono K. Modulation of microRNA processing by p53. Nature. 2009;460: 529 – 533. 33. Mine N, Kurose K, Nagai H, Doi D, O ta Y, Yone yama K, Konishi H, Araki T, Emi M. Gene fusion involving HMGIC is a frequent aberration in uterine leiomyomas. J Hum Genet. 2001;46:408 – 412. 34. Martin Chave s EB, Brum IS, Stoll J, Capp E, Corle ta HE. Insulin-like growth factor 1 receptor mRNA expression and autophosphoryla- tion in human myometrium and leiomyoma. Gynecol Obstet Invest. 2004;57:210 –213. 35. Gao Z, Matsuo H, Wang Y, Nakago S, Maruo T. Up-regulation by IGF-I of proliferating cell nuclear antigen and Bcl-2 protein expression in human uterine leiomyoma cells. J Clin Endocrinol Metab. 2001;86:5593–5599. 36. Pe ng Y, Lase r J, Shi G, Mittal K, Me lame d J, Le e P, We i JJ. Anti- proliferative effects by Let-7 repression of high-mobility group A2 in uterine leiomyoma. Mol Cancer Res. 2008;6:663– 673. 37. Markowski DN, Winte r N, Me ye r F, von Ahse n I, We nk H, Nolte I, Bulle rdie k J. p14Arf acts as an antagonist of HMGA2 in senescence of mesenchymal stem cells-implications for benign tumorigenesis. Genes Chromosomes Cancer. 2011;50:489 – 498. 38. Yu KR, Park SB, Jung JW, Se o MS, Hong IS, Kim HS, Se o Y, Kang TW, Le e JY, Kurtz A, Kang KS. HMGA2 regulates the in vitro aging and proliferation of human umbilical cord blood-derived stromal cells through the mT OR/p70S6K signaling pathway. Stem cell re- search. 2013;10:156 – 165.

10