Introduction of Somatic Mutation in MED12 Induces Wnt4/ß-Catenin and Disrupts Autophagy in Human Uterine Myometrial Cell.

Uterine fibroids (UFs) or leiomyoma are frequently associated with somatic mutations in the mediator complex subunit 12 (MED12) gene; however, the function of these mutations in human UF biology is yet to be determined. Herein, we determined the functional role of the most common MED12 somatic mutation in the modulation of oncogenic Wnt4/ß-catenin and mammalian target of rapamycin (mTOR) signaling pathways. Using an immortalized human uterine myometrial smooth muscle cell line (UtSM), we constitutively overexpressed either MED12-Wild Type or the most common MED12 somatic mutation (c.131G>A), and the effects of this MED12 mutation were compared between these cell lines. This immortalized cell line was used as a model because it expresses wild type MED12 protein and do not possess MED12 somatic mutations. By comparing the effect between MED12-WT and MED12-mutant (mut) stable cell populations, we observed increased levels of protein expression of Wnt4 and ß-catenin in MED12-mut cells as compared with MED12-WT cells. MED12-mut cells also expressed increased levels of mTOR protein and oncogenic cyclin D1 which are hallmarks of cell growth and tumorigenicity. This somatic mutation in MED12 showed an effect on cell-cycle progression by induction of S-phase cells. MED12-mut cells also showed inhibition of autophagy as compared with MED12-WT cells. Together, these findings indicate that the MED12 somatic mutation has the potentials for myometrial cell transformation by dysregulating oncogenic Wnt4/ß-catenin and its downstream mTOR signaling which might be associated with autophagy abrogation, cell proliferation, and tumorigenicity.

Mediator Kinase Disruption in MED12-Mutant Uterine Fibroids From Hispanic Women of South Texas.

Context: Mutations in the gene encoding Mediator complex subunit MED12 are dominant drivers of uterine fibroids (UFs) in women of diverse racial and ethnic origins. Previously, we showed that UF-linked mutations in MED12 disrupt its ability to activate cyclin C-CDK8/19 in Mediator. However, validation of Mediator kinase disruption in the clinically relevant setting of MED12-mutant UFs is currently lacking. Objective: The objective of this study was twofold. First, to extend the ethnic distribution profile of MED12 mutations by establishing their frequency in UFs from Hispanic women of South Texas. Second, to examine the impact of MED12 mutations on Mediator kinase activity in patient-derived UFs. Methods: We screened 219 UFs from 76 women, including 170 tumors from 57 Hispanic patients, for MED12 exon 2 mutations, and further examined CDK8/19 activity in Mediator complexes immunoprecipitated from MED12 mutation-negative and MED12 mutation-positive UFs. Results: MED12 exon 2 mutations in UFs from Hispanic women are somatic in nature, predominantly monoallelic, and occur at high frequency (54.1%). We identified a minimal cyclin C-CDK8 activation domain on MED12 spanning amino acids 15 through 80 that includes all recorded UF-linked mutations in MED12, suggesting that disruption of Mediator kinase activity is a principal biochemical defect arising from these pathogenic alterations. Analysis of Mediator complexes recovered from patient UFs confirmed this, revealing that Mediator kinase activity is selectively impaired in MED12-mutant UFs. Conclusions: MED12 mutations are important drivers of UF formation in Hispanic women of South Texas. MED12 mutations disrupt Mediator kinase activity, implicating altered CDK8/19 function in UF pathogenesis.

Oncogenic exon 2 mutations in Mediator subunit MED12 disrupt allosteric activation of cyclin C-CDK8/19.

Somatic mutations in exon 2 of the RNA polymerase II transcriptional Mediator subunit MED12 occur at high frequency in uterine fibroids (UFs) and breast fibroepithelial tumors as well as recurrently, albeit less frequently, in malignant uterine leimyosarcomas, chronic lymphocytic leukemias, and colorectal cancers. Previously, we reported that UF-linked mutations in MED12 disrupt its ability to activate cyclin C (CycC)-dependent kinase 8 (CDK8) in Mediator, implicating impaired Mediator-associated CDK8 activity in the molecular pathogenesis of these clinically significant lesions. Notably, the CDK8 paralog CDK19 is also expressed in myometrium, and both CDK8 and CDK19 assemble into Mediator in a mutually exclusive manner, suggesting that CDK19 activity may also be germane to the pathogenesis of MED12 mutation-induced UFs. However, whether and how UF-linked mutations in MED12 affect CDK19 activation is unknown. Herein, we show that MED12 allosterically activates CDK19 and that UF-linked exon 2 mutations in MED12 disrupt its CDK19 stimulatory activity. Furthermore, we find that within the Mediator kinase module, MED13 directly binds to the MED12 C terminus, thereby suppressing an apparent UF mutation-induced conformational change in MED12 that otherwise disrupts its association with CycC-CDK8/19. Thus, in the presence of MED13, mutant MED12 can bind, but cannot activate, CycC-CDK8/19. These findings indicate that MED12 binding is necessary but not sufficient for CycC-CDK8/19 activation and reveal an additional step in the MED12-dependent activation process, one critically dependent on MED12 residues altered by UF-linked exon 2 mutations. These findings confirm that UF-linked mutations in MED12 disrupt composite Mediator-associated kinase activity and identify CDK8/19 as prospective therapeutic targets in UFs.

Silencing Med12 Gene Reduces Proliferation of Human Leiomyoma Cells Mediated via Wnt/ß-Catenin Signaling Pathway.

Uterine fibroids, or leiomyoma, are the most common benign tumors in women of reproductive age. In this work, the effect of silencing the mediator complex subunit 12 (Med12) gene in human uterine fibroid cells was evaluated. The role of Med12 in the modulation of Wnt/ß-catenin and cell proliferation-associated signaling was evaluated in human uterine fibroid cells. Med12 was silenced in the immortalized human uterine fibroid cell line (HuLM) using a lentivirus-based Med12 gene-specific RNA interference strategy. HuLM cells were infected with lentiviruses carrying Med12-specific short hairpin RNA (shRNA) sequences or a nonfunctional shRNA scrambled control with green fluorescence protein. Stable cells that expressed low levels of Med12 protein were characterized. Wnt/ß-catenin signaling, sex steroid receptor signaling, cell cycle-associated, and fibrosis-associated proteins were measured. Med12 knockdown cells showed significantly (P < 0.05) reduced levels of Wnt4 and ß-catenin proteins as well as cell proliferation, as compared with scrambled control cells. Med12 knockdown cells also showed reduced levels of cell cycle-associated cyclin D1, Cdk1, and Cdk2 proteins as well as reduced activation of p-extracellular signal-regulated kinase, p-protein kinase B, and transforming growth factor (TGF)-ß signaling pathways as compared with scrambled control cells. Moreover, TGF-ß-regulated fibrosis-related proteins such as fibronectin, collagen type 1, and plasminogen activator inhibitor-1 were significantly (P < 0.05) reduced in Med12 knockdown cells as compared with scrambled control cells. Together, these results suggest that Med12 plays a key role in the regulation of HuLM cell proliferation through the modulation of Wnt/ß-catenin, cell cycle-associated, and fibrosis-associated protein expression.

Vitamin D3 Inhibits Wnt/ß-Catenin and mTOR Signaling Pathways in Human Uterine Fibroid Cells.

CONTEXT: Somatic mutations in the Med12 gene are known to activate Wnt/ß-catenin signaling in human uterine fibroids (UFs). OBJECTIVE: The objective of the study was to examine the role of vitamin D3 in the modulation of Wnt/ß-catenin and mammalian target of rapamycin (mTOR) signaling in human UF cells. DESIGN: Immortalized human UF cells (HuLM) and human primary UF (PUF) cells were treated with increasing concentrations of vitamin D3 and thereafter analyzed using Western blots and immunocytochemistry. MAIN OUTCOME MEASURES: Wnt/ß-catenin and mTOR signaling proteins in cultured HuLM and PUF cells were measured. RESULTS: UF tumors with Med12 somatic mutations showed an up-regulation of Wnt4 and ß-catenin as compared with adjacent myometrium. Vitamin D3 administration reduced the levels of Wnt4 and ß-catenin in both HuLM and PUF cells. Vitamin D3 also reduced the expression/activation of mTOR signaling in both cell types. In contrast, vitamin D3 induced the expression of DNA damaged-induced transcription 4 (an inhibitor of mTOR) and tuberous sclerosis genes (TSC1/2) in a concentration-dependent manner in HuLM cells. Furthermore, we observed a concentration-dependent reduction of Wisp1 (Wnt induced signaling protein 1) and flap endonuclease 1 proteins in HuLM cells. Additionally, abrogation of vitamin D receptor expression (by silencing) in normal myometrial cells induces Wnt4/ß-catenin as well as prompts a fibrotic process including an increase in cell proliferation and increased extracellular matrix production. Together these results suggest that vitamin D3 functions as an inhibitor of Wnt4/ß-catenin and mTOR signaling pathways, which may play major roles in fibroid pathogenesis. CONCLUSIONS: Vitamin D3 may have utility as a novel long-term therapeutic and/or preventive option for uterine fibroids.

Role of vitamin D in uterine fibroid biology.

OBJECTIVE: To provide a detailed summary of current scientific knowledge on uterine fibroids (leiomyomas) in vitro and in in vivo animal models, as well as to postulate the potential role of vitamin D3 as an effective, inexpensive, safe, long-term treatment option for uterine fibroids. DESIGN: PubMed search articles were used to identify the most relevant studies on uterine fibroids, as well as effects of vitamin D3 on uterine fibroid cells and fibroid tumor growth in in vivo animal models. SETTING: University research laboratory. PATIENT(S): Not applicable. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Not applicable. RESULT(S): Despite numerous publications available on uterine fibroids, information about the role that vitamin D3 plays in the regulation of uterine fibroids is limited. Most of the recent vitamin D3-related studies on uterine fibroids were published from our group. Recent studies have demonstrated that vitamin D deficiency plays a significant role in the development of uterine fibroids. Our recent studies have demonstrated that vitamin D3 reduces leiomyoma cell proliferation in vitro and leiomyoma tumor growth in in vivo animal models. These results postulate the potential role of vitamin D3 for an effective, safe, nonsurgical medical treatment option for uterine fibroids. CONCLUSION(S): This article reviews human and animal studies and uncovers new possibilities for understanding the vitamin D-based therapeutic option for an effective, safe, long-term treatment of uterine fibroids. On the basis of these results, a clinical trial with vitamin D3 or a hypocalcemic analog, paricalcitol, may be warranted for nonsurgical medical treatment of uterine fibroids.

1,25-dihydroxyvitamin D3 regulates expression of sex steroid receptors in human uterine fibroid cells.

CONTEXT: Uterine fibroids (UFs) are the most common benign tumors in premenopausal women. In this study, we evaluated the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] for the treatment of UFs. OBJECTIVE: To determine the role of 1,25(OH)2D3 on the expression of sex steroid receptors in human UF cells. DESIGN: Human UFs and their adjacent myometrium were analyzed for expression of estrogen receptor (ER)-α, progesterone receptor (PR)-A, and PR-B, as well as members of the steroid receptor coactivator (SRC) family. Immortalized human uterine fibroid (human uterine leiomyoma [HuLM]) cells were treated with 1,25(OH)2D3 and assayed for the expression and localization of the aforementioned receptors and SRCs using Western blot, immunohistochemistry, immunofluorescence, and immunoprecipitation assays. MAIN OUTCOME MEASURES: We discovered a correlation between reduced levels of vitamin D receptor (VDR) and increased levels of ER-α, PR-A, and PR-B in these tissues. We evaluated the effects of 1,25(OH)2D3 on the regulation of the aforementioned sex steroid receptors. RESULTS: We observed an inverse correlation between the up-regulated ER-α, PR-A, and PR-B and expression of VDR in UFs. Treatment with 1,25(OH)2D3 significantly decreased levels of ER-α, PR-A, and PR-B, as well as SRCs in HuLM cells (P < .05). In contrast, 1,25(OH)2D3 self-induced its own VDR, which resulted in an induction of VDR-retinoid X receptor-α complex in HuLM cells. Together, these results suggest that 1,25(OH)2D3 functions as an antagonist of sex steroid hormone receptors in HuLM cells. CONCLUSIONS: 1,25(OH)2D3 functions as a potent antiestrogenic/antiprogesteronic agent that may have utility as a novel therapeutic option for UF.

Novel MED12 gene somatic mutations in women from the Southern United States with symptomatic uterine fibroids.

Although somatic mutations in exon 2 of the mediator complex subunit 12 (MED12) gene have been reported previously in uterine fibroids in women from Finland, South Africa, and North America, the status of these mutations was not reported in the Southern United States women. The aim of this study is to determine the MED12 somatic mutations in uterine fibroids of women from Southern Unites States, which will help to better understand the contribution of MED12 mutations in fibroid tumor biology. Herein, we determined the frequency of MED12 gene exon 2 somatic mutations in 143 fibroid tumors from a total of 135 women from the Southern United States and in 50 samples of the adjacent myometrium using PCR amplification and Sanger sequencing. We observed that the MED12 gene is mutated in 64.33 % (92/143) of uterine fibroid cases in the exon 2 (including deletion mutations). These mutations include 107T > G (4.3 %), 130G > C (2.8 %), 130G > A (7.0 %), 130G > T (2.8 %), 131G > C (2.1 %), 131G > A (20.2 %), and 131G > T (2.1 %). Interestingly, we identified four novel mutations in these patients: 107 T > C (12.8 %), 105A > T (2.1 %), 122T > A (2.1 %), and 92T > A (2.1 %). As expected, we did not observe any mutations in the normal myometrium. Moreover, we found a higher rate of deletion mutations (17.5 %, 25/143) in the above fibroid tumors. Our results clearly demonstrate that the MED12 gene exon 2 is frequently mutated in human uterine fibroids in Southern United States women. These results highlight the molecular pathogenesis of human uterine fibroids with the central role of MED12 somatic mutations.

Paricalcitol, a vitamin d receptor activator, inhibits tumor formation in a murine model of uterine fibroids.

We examined the antitumor and therapeutic potentials of paricalcitol, an analog of 1,25-dihydroxyvitamin D3 with lower calcemic activity, against uterine fibroids using in vitro and in vivo evaluations in appropriate uterine fibroid cells and animal models. We found that paricalcitol has potential to reduce the proliferation of the immortalized human uterine fibroid cells. For the in vivo study, we generated subcutaneous tumors by injecting the Eker rat-derived uterine leiomyoma cell line (ELT-3) rat uterine fibroid-derived cell line in athymic nude mice supplemented with estrogen pellets. These mice were administered with vehicle versus paricalcitol (300 ng/kg/d) or 1,25-dihydroxyvitamin D3 (500 ng/kg/d) for 4 consecutive weeks, and the data were analyzed. We found that while both paricalcitol and 1,25-dihydroxyvitamin D3 significantly reduced fibroid tumor size, the shrinkage was slightly higher in the paricalcitol-treated group. Together, our results suggest that paricalcitol may be a potential candidate for effective, safe, and noninvasive medical treatment option for uterine fibroids.

1,25-dihydroxyvitamin d3 reduces extracellular matrix-associated protein expression in human uterine fibroid cells.

Uterine fibroids (leiomyomas) are the most common benign tumors associated with excessive deposition of extracellular matrix (ECM)-associated proteins that increase fibroid tumorigenicity. Herein, we determined the expression levels of vitamin D receptor (VDR) protein in human uterine fibroids and compared these levels to those in adjacent normal myometrium. Using Western blot analysis, we found that more than 60% of uterine fibroids analyzed (25 of 40) expressed low levels of VDR. We also found that the biologically active 1,25-dihydroxyvitamin D3 (1,25[OH]2D3), which functions via binding to its nuclear VDR, induced VDR in a concentration-dependent manner and reduced ECM-associated fibrotic and proteoglycans expression in immortalized human uterine fibroid cell line (HuLM). At 1-10 nM concentrations, 1,25(OH)2D3 significantly induced (P < 0.05) nuclear VDR, which was further stimulated by higher concentrations of 1,25(OH)2D3 in HuLM cells. 1,25(OH)2D3 at 10 nM also significantly reduced (P < 0.05) the protein expression of ECM-associated collagen type 1, fibronectin, and plasminogen activator inhibitor-1 (PAI-1) in HuLM cells. We also found that 1,25(OH)2D3 reduced mRNA and protein expressions of proteoglycans such as fibromodulin, biglycan, and versican in HuLM cells. Moreover, the aberrant expression of structural smooth muscle actin fibers was reduced by 1,25(OH)2D3 treatment in a concentration-dependent manner in HuLM cells. Taken together, our results suggest that human uterine fibroids express reduced levels of VDR compared to the adjacent normal myometrium and that treatment with 1,25(OH)2D3 can potentially reduce the aberrant expression of major ECM-associated proteins in HuLM cells. Thus, 1,25(OH)2D3 might be an effective, safe, nonsurgical treatment option for human uterine fibroids.

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.

Serum vitamin D3 level inversely correlates with uterine fibroid volume in different ethnic groups: a cross-sectional observational study.

PURPOSE: Currently there is no effective medicinal treatment for uterine fibroids (UFs), a common health disorder that affects women of reproductive age. Identification of modifiable risk factors such as vitamin D (Vit D) deficiency could help develop novel strategies for the prevention and/or treatment of UFs. The purpose of this study was to identify whether low serum Vit D3 levels correlate with increased risk of UFs. METHODS: A total of 154 premenopausal women were recruited for this cross-sectional study. The control group comprised 50 subjects with a normal, fibroid-free uterine structure, confirmed by transvaginal ultrasonography. The 104 case subjects had at least one fibroid lesion that was 2 cm(3) in volume or larger, confirmed by transvaginal ultrasonography. For each case subject, total uterine volume and total volume of all existing fibroids were measured in three perpendicular planes, with volume determined according to the prolate ellipse formula (a × b × c × 0.523), where a is height, b is width, and c is depth. Serum Vit D [25(OH) D3] levels were measured by radioimmunoassay. The independent t-test was used to compare serum Vit D levels across groups. Correlations were assessed by Spearman's rank correlation test. RESULTS: Lower serum 25-(OH) Vit D levels were significantly associated with the occurrence of UFs (P = 0.01). A statistically significant inverse correlation was also observed between serum 25-(OH) Vit D levels and total UF volume (r = -0.31; P = 0.002) within the case cohort. Subjects with larger fibroid volumes had lower serum Vit D levels and vice versa. Data stratified for ethnicity showed a statistically significant inverse correlation between serum 25-(OH) Vit D levels and total fibroid volume in black subjects (r = -0.42; P = 0.001). An inverse correlation was also evident in white subjects (r = -0.86; P = 0.58) but this did not reach statistical significance. CONCLUSION: Lower serum Vit D levels are inversely correlated with UF burden in different ethnic groups. Vit D deficiency is a possible risk factor for the occurrence of UFs.

Role of TGF-ß signaling in curcumin-mediated inhibition of tumorigenicity of human lung cancer cells.

PURPOSE: Curcumin has been shown to have potent anticancer activities like inhibition of cell proliferation, induction of apoptosis, and suppression of angiogenesis. Transforming growth factor-ß (TGF-ß) signaling plays a complex role in tumor suppression and promotion depending on the tumor type and stage. However, the effect of curcumin on TGF-ß signaling in cancer cells and the role of TGF-ß signaling in curcumin-induced anticancer activities have not been determined. Here, we investigate the role of curcumin on TGF-ß signaling, and whether TGF-ß signaling is involved in the antitumor activities of curcumin. METHODS: Human non-small cell lung cancer (NSCLC) cell lines, ACC-LC-176 (without TGF-ß signaling), H358, and A549 (with TGF-ß signaling) were treated with curcumin to determine cell growth, apoptosis, and tumorigenicity. Antitumor activities of curcumin were determined using these cell lines and an in vivo mouse model. We also tested the effect of curcumin on TGF-ß/Smad signaling by western blotting and by luciferase assays. RESULTS: Curcumin inhibited cell growth and induced apoptosis of all three NSCLC cell lines in vitro and in vivo. It significantly reduced subcutaneous tumor growth by these three cell lines irrespective of TGF-ß signaling status. Curcumin inhibited TGF-ß-induced Smad2/3 phosphorylation and transcription in H358 and A549 cells, but not in ACC-LC-176 cells. CONCLUSIONS: Curcumin reduces tumorigenicity of human lung cancer cells in vitro and in vivo by inhibiting cell proliferation and promoting apoptosis. These results suggest that TGF-ß signaling is not directly involved in curcumin-mediated growth inhibition, induction of apoptosis, and inhibition of tumorigenicity.

1,25-dihydroxyvitamin D3 treatment shrinks uterine leiomyoma tumors in the Eker rat model.

Uterine leiomyomas (fibroids) are the most common benign tumors in women of reproductive age. These tumors are three to four times more prevalent in African American women, who also have a 10 times higher incidence of hypovitaminosis D than white women. Recent studies have demonstrated the antitumor effects of 1,25-dihydroxyvitamin D3 on several cancers, but its effects on uterine leiomyomas are still unknown. To determine the antitumor and therapeutic effects of 1,25-dihydroxyvitamin D3 on uterine leiomyomas, female Eker rats (14-16 mo old) harboring uterine leiomyomas were randomized into control and experimental groups and were given vehicle versus 1,25-dihydroxyvitamin D3 (0.5 µg/kg per day) subcutaneously for 3 wk, respectively. At the end of the experiment, the rats were euthanized, and the leiomyoma tumors were analyzed. Treatment with 1,25-dihydroxyvitamin D3 significantly reduced leiomyoma tumor size in Eker rats. It also reduced leiomyoma size by suppressing cell growth and proliferation-related genes (Pcna, cyclin D1 [Ccnd1], Myc, Cdk1, Cdk2, and Cdk4), antiapoptotic genes (Bcl2 and Bcl2l1 [Bcl-x]), and estrogen and progesterone receptors. Additionally, immunohistochemistry revealed decreased expression of PCNA and MKI67 (a marker of proliferation) and increased expression of caspase 3 in 1,25-dihydroxyvitamin D3-treated Eker rat leiomyomas. Toxicity analyses using serum samples showed similar levels of SGOT, SGPT, calcium, and total bilirubin in 1,25-dihydroxyvitamin D3-treated and vehicle-treated control Eker rats. These results support that 1,25-dihydroxyvitamin D3 is an antitumor agent that may be a potential safe, nonsurgical therapeutic option for the treatment of uterine leiomyomas.

Elucidating the mechanism of regulation of transforming growth factor ß Type II receptor expression in human lung cancer cell lines.

Lung carcinogenesis in humans involves an accumulation of genetic and epigenetic changes that lead to alterations in normal lung epithelium, to in situ carcinoma, and finally to invasive and metastatic cancers. The loss of transforming growth factor ß (TGF-ß)-induced tumor suppressor function in tumors plays a pivotal role in this process, and our previous studies have shown that resistance to TGF-ß in lung cancers occurs mostly through the loss of TGF-ß type II receptor expression (TßRII). However, little is known about the mechanism of down-regulation of TßRII and how histone deacetylase (HDAC) inhibitors (HDIs) can restore TGF-ß-induced tumor suppressor function. Here we show that HDIs restore TßRII expression and that DNA hypermethylation has no effect on TßRII promoter activity in lung cancer cell lines. TGF-ß-induced tumor suppressor function is restored by HDIs in lung cancer cell lines that lack TßRII expression. Activation of mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by either activated Ras or epidermal growth factor signaling is involved in the down-regulation of TßRII through histone deacetylation. We have immunoprecipitated the protein complexes by biotinylated oligonucleotides corresponding to the HDI-responsive element in the TßRII promoter (-127/-75) and identified the proteins/factors using proteomics studies. The transcriptional repressor Meis1/2 is involved in repressing the TßRII promoter activity, possibly through its recruitment by Sp1 and NF-YA to the promoter. These results suggest a mechanism for the downregulation of TßRII in lung cancer and that TGF-ß tumor suppressor functions may be restored by HDIs in lung cancer patients with the loss of TßRII expression.

1,25-Dihydroxyvitamin D3 reduces TGF-beta3-induced fibrosis-related gene expression in human uterine leiomyoma cells.

BACKGROUND: Uterine leiomyomas (fibroids) are the most common benign estrogen-dependent tumors of premenopausal women. TGF-ß3 up-regulates the synthesis of many of extracellular matrix proteins that are associated with tissue fibrosis. OBJECTIVE: To examine the effect of 1,25-dihydroxyvitamin D(3) (vitamin D(3)) on TGF-ß3-induced fibrosis-related protein expression in immortalized human uterine leiomyoma (HuLM) cells. METHODS: HuLM cells were treated with TGF-ß3 with or without vitamin D(3). Western blot analyses were employed to test the effect of vitamin D(3) on TGF-ß3-induced protein expression of collagen type 1, fibronectin, and plasminogen activator inhibitor-1 proteins. Western blots as well as immunofluorescence analyses were used to verify the effect of vitamin D(3) on TGF-ß3-induced Smad activation involved in extracellular matrix protein synthesis and deposition, which ultimately lead to tissue fibrosis. RESULTS: We observed that TGF-ß3 induced fibronectin and collagen type 1 protein expression in HuLM cells, and that effect was suppressed by vitamin D(3). TGF-ß3 also induced protein expression of plasminogen activator inhibitor-1, an important TGF-ß target, in HuLM cells, which was also inhibited by vitamin D(3). Additionally, TGF-ß3 induced phosphorylation of Smad2 as well as nuclear translocation of Smad2 and Smad3 in HuLM cells, whereas vitamin D significantly reduced all these TGF-ß3-mediated effects. Therefore, our results suggest that vitamin D(3) has consistently reduced TGF-ß3 effects that are involved in the process of fibrosis in human leiomyoma cells. CONCLUSION: Vitamin D(3) is an antifibrotic factor that might be potentially useful as a novel therapeutic for nonsurgical treatment of benign uterine fibroids.

Vitamin D inhibits proliferation of human uterine leiomyoma cells via catechol-O-methyltransferase.

OBJECTIVE: To evaluate the effects and mechanisms of action of vitamin D on human uterine leiomyoma (HuLM) cell proliferation in vitro. DESIGN: Laboratory study. SETTING: University hospitals. PATIENTS(S): Not applicable. INTERVENTIONS(S): Not applicable. MAIN OUTCOME MEASURE(S): HuLM cells were treated with 1,25-dihydroxyvitamin D3 (vitamin D), and cell proliferation was assayed by the methylthiazolyl tetrazolium technique. proliferating cell nuclear antigen (PCNA), BCL-2, BCL-w, cyclin-dependent kinase (CDK) 1, and catechol-O-methyltransferase (COMT) protein levels were analyzed by Western blotting. COMT mRNA and enzyme activity were assayed by quantitative reverse-transcription polymerase chain reaction (RT-PCR) and high-performance liquid chromatography analysis, respectively. The role of COMT was evaluated in stable HuLM cells by silencing COMT expression. RESULT(S): Vitamin D inhibited the growth of HuLM cells by 47±0.03% at 1 µM and by 38±0.02% at 0.1 µM compared with control cells at 120 hours of treatment. Vitamin D inhibited extracellular signal-regulated kinase activation and down-regulated the expression of BCL-2, BCL-w, CDK1, and PCNA. Western blot, RT-PCR, and enzyme assay of COMT demonstrated inhibitory effects of vitamin D on COMT expression and enzyme activity. Silencing endogenous COMT expression abolished vitamin D-mediated inhibition of HuLM cell proliferation. CONCLUSION(S): Vitamin D inhibits growth of HuLM cells through the down-regulation of PCNA, CDK1, and BCL-2 and suppresses COMT expression and activity in HuLM cells. Thus, hypovitaminosis D appears to be a risk factor for uterine fibroids.

Antimetastatic role of Smad4 signaling in colorectal cancer.

BACKGROUND & AIMS: Transforming growth factor (TGF)-beta signaling occurs through Smads 2/3/4, which translocate to the nucleus to regulate transcription; TGF-beta has tumor-suppressive effects in some tumor models and pro-metastatic effects in others. In patients with colorectal cancer (CRC), mutations or reduced levels of Smad4 have been correlated with reduced survival. However, the function of Smad signaling and the effects of TGF-beta-receptor kinase inhibitors have not been analyzed during CRC metastasis. We investigated the role of TGF-beta/Smad signaling in CRC progression. METHODS: We evaluated the role of TGF-beta/Smad signaling on cell proliferation, migration, invasion, tumorigenicity, and metastasis in Smad4-null colon carcinoma cell lines (MC38 and SW620) and in those that transgenically express Smad4. We also determined the effects of a TGF-beta-receptor kinase inhibitor (LY2109761) in CRC tumor progression and metastasis in mice. RESULTS: TGF-beta induced migration/invasion, tumorigenicity, and metastasis of Smad4-null MC38 and SW620 cells; incubation with LY2109761 reversed these effects. In mice, LY2109761 blocked metastasis of CRC cells to liver, inducing cancer cell expression of E-cadherin and reducing the expression of the tumorigenic proteins matrix metalloproteinase-9, nm23, urokinase plasminogen activator, and cyclooxygenase-2. Transgenic expression of Smad4 significantly reduced the oncogenic potential of MC38 and SW620 cells; in these transgenic cells, TGF-beta had tumor suppressor, rather than tumorigenic, effects. CONCLUSIONS: TGF-beta/Smad signaling suppresses progression and metastasis of CRC cells and tumors in mice. Loss of Smad4 might underlie the functional shift of TGF-beta from a tumor suppressor to a tumor promoter; inhibitors of TGF-beta signaling might be developed as CRC therapeutics.

Targeting transforming growth factor-beta signaling in liver metastasis of colon cancer.

Despite a primary tumor suppressor role, there is compelling evidence suggesting that TGF-beta can promote tumor growth, invasion and metastasis in advanced stages of colorectal cancer. Blocking these tumor-promoting effects of TGF-beta provides a potentially important therapeutic strategy for the treatment of colorectal cancer. However, little is known about how the inhibitors of TGF-beta receptor kinases affect colorectal carcinogenesis in vivo. Here, we have observed that a novel dual kinase inhibitor of TGF-beta type I and type II receptors, LY2109761, inhibits TGF-beta-mediated activation of Smad and non-Smad pathways in CT26 colon adenocarcinoma cells having K-Ras mutation. The inhibitor attenuates the oncogenic effects of TGF-beta on cell migration, invasion and tumorigenicity of CT26 cells. Furthermore, LY2109761 decreases liver metastases and prolongs survival in an experimental metastasis model. These findings suggest that the dual kinase inhibitor LY2109761 has potential therapeutic value for metastatic colorectal cancer.

Oncogenic serine-threonine kinase receptor-associated protein modulates the function of Ewing sarcoma protein through a novel mechanism.

Although much is known about the oncogenic functions of chimeric Ewing sarcoma (EWS) fusion proteins that result from chromosomal translocations, the cellular role of the normal EWS protein is not well characterized. We have previously identified a WD domain-containing protein, serine-threonine kinase receptor-associated protein (STRAP), which inhibits transforming growth factor beta (TGF-beta) signaling through interaction with receptors and Smad7 and promotes growth and enhances tumorigenicity. Here, we report the interaction between STRAP and EWS using matrix-assisted laser desorption/ionization, time-of-flight and tandem mass spectrometry. Although STRAP is localized in both cytoplasm and nucleus, nuclear STRAP colocalizes and associates specifically with EWS in the nucleus through its NH(2) and COOH termini. We have found that normal EWS protein is up-regulated in human cancers, which correlates with the up-regulation of STRAP in 71% of colorectal cancers and 54% of lung cancers, suggesting a cooperative role of these two proteins in human cancers. TGF-beta has no effect on STRAP and EWS interaction. However, EWS, like STRAP, attenuates TGF-beta-dependent transcription. STRAP inhibits EWS-dependent p300-mediated transactivation of EWS target genes, such as ApoCIII and c-fos, in a TGF-beta-independent manner. Interestingly, we have shown that STRAP blocks the interaction between EWS and p300, whereas the complex formation between STRAP and EWS is not affected by p300. These results suggest that STRAP inhibits the transactivation function of EWS by displacing p300 from the functional transcriptional complex. Thus, this study provides a novel TGF-beta-independent function of STRAP and describes a mechanism by which STRAP regulates the function of oncogenic EWS protein.