SIX1 cooperates with RUNX1 and SMAD4 in cell fate commitment of Müllerian duct epithelium.

During female mammal reproductive tract development, epithelial cells of the lower Müllerian duct are committed to become stratified squamous epithelium of the vagina and ectocervix, when the expression of ΔNp63 transcription factor is induced by mesenchymal cells. The absence of ΔNp63 expression leads to adenosis, the putative precursor of vaginal adenocarcinoma. Our previous studies with genetically engineered mouse models have established that fibroblast growth factor (FGF)/mitogen-activated protein kinase (MAPK), bone morphogenetic protein (BMP)/SMAD, and activin A/runt-related transcription factor 1 (RUNX1) signaling pathways are independently required for ΔNp63 expression in Müllerian duct epithelium (MDE). Here, we report that sine oculis homeobox homolog 1 (SIX1) plays a critical role in the activation of ΔNp63 locus in MDE as a downstream transcription factor of mesenchymal signals. In the developing mouse reproductive tract, SIX1 expression was restricted to MDE within the future cervix and vagina. SIX1 expression was totally absent in SMAD4 null MDE and was reduced in RUNX1 null and FGFR2 null MDE, indicating that SIX1 is under the control of vaginal mesenchymal factors: BMP4, activin A and FGF7/10. Furthermore, Six1, Runx1, and Smad4 gene-dose-dependently activated ΔNp63 expression in MDE within the vaginal fornix. Using a mouse model of diethylstilbestrol (DES)-associated vaginal adenosis, we found DES action through epithelial estrogen receptor α (ESR1) inhibits activation of ΔNp63 locus in MDE by transcriptionally repressing SIX1 and RUNX1 in the vaginal fornix.

Transient inhibition of p53 homologs protects ovarian function from two distinct apoptotic pathways triggered by anticancer therapies.

Platinum-based chemotherapies can result in ovarian insufficiency by reducing the ovarian reserve, a reduction believed to result from apoptosis of immature oocytes via activation/phosphorylation of TAp63α by multiple kinases including CHEK2, CK1, and ABL1. Here we demonstrate that cisplatin (CDDP) induces oocyte apoptosis through a novel pathway and that temporary repression of this pathway fully preserves ovarian function in vivo. Although ABL kinase inhibitors effectively block CDDP-induced apoptosis of oocytes, oocytic ABL1, and ABL2 are dispensable for damage-induced apoptosis. Instead, CDDP activates TAp63α through the ATR > CHEK1 pathway independent of TAp63α hyper-phosphorylation, whereas X-irradiation activates the ATM > CHEK2 > TAp63α-hyper-phosphorylation pathway. Furthermore, oocyte-specific deletion of Trp73 partially protects oocytes from CDDP but not from X-ray, highlighting the fundamental differences of two pathways. Nevertheless, temporary repression of DNA damage response by a kinase inhibitor that attenuates phosphorylation of ATM, ATR, CHEK1, and CHEK2 fully preserves fertility in female mice against CDDP as well as X-ray. Our current study establishes the molecular basis and feasibility of adjuvant therapies to protect ovarian function against two distinctive gonadotoxic therapeutics, CDDP, and ionizing radiation.

Cellular kinetics of MED12-mutant uterine leiomyoma growth and regression in vivo.

Cellular mechanisms of uterine leiomyoma (LM) formation have been studied primarily utilizing in vitro models. However, recent studies established that the cells growing in the primary cultures of MED12-mutant LM (MED12-LM) do not carry causal mutations. To improve the accuracy of LM research, we addressed the cellular mechanisms of LM growth and regression utilizing a patient-derived xenograft (PDX) model, which faithfully replicates the patient tumors in situ The growth and maintenance of MED12-LMs depend on 17ß-estradiol (E2) and progesterone (P4). We determined E2 and P4-activated MAPK and PI3K pathways in PDXs with upregulation of IGF1 and IGF2, suggesting that the hormone actions on MED12-LM are mediated by the IGF pathway. When hormones were removed, MED12-LM PDXs lost approximately 60% of volume within 3 days through reduction in cell size. However, in contrast to general belief, the survival of LM cells was independent of E2 and/or P4, and apoptosis was not involved in the tumor regression. Furthermore, it was postulated that abnormal collagen fibers promote the growth of LMs. However, collagen fibers of actively growing PDXs were well aligned. The disruption of collagen fibers, as found in human LM specimens, occurred only when the volume of PDXs had grown to over 20 times the volume of unstimulated PDXs, indicating disruption is the result of growth not the cause. Hence, this study revises generally accepted theories on the growth and regression of LMs.

Novel SOX17 frameshift mutations in endometrial cancer are functionally distinct from recurrent missense mutations.

Extensive genomic profiling for endometrioid endometrial carcinoma (EEC) has pointed to genes and pathways important in uterine development as critical mediators of endometrial tumorigenesis. SOX17 is a developmental transcription factor necessary for proper endoderm formation that has been implicated as a tumor suppressor and shown to modulate WNT signaling. SOX17 mutation analysis in 539 primary EECs revealed frequent missense and frameshift mutations with an overall 11.5% mutation rate. More than half the mutations identified were frameshifts (32 of 62), and the hotspot missense changes, p.Ala96Gly and p.Ser403Ile, were seen in 14 tumors. None of the cases with a mutation had a second SOX17 mutation or evidence of allelic loss. Immunofluorescence microscopy performed on primary samples showed that there were no changes in SOX17 protein expression associated with mutation. Low/absent SOX17 staining was significantly associated with advanced stage, high tumor grade and reduced recurrence-free survival. Functional assessment of the two hotspot missense mutations and three representative frameshift mutations showed that SOX17-A96G and SOX17-S403I have transcriptional activities similar to SOX17 wild-type (WT), whereas none of the frameshift mutant proteins showed transcriptional activity. Forced expression of SOX17-WT, -A96G or -S403I in EC cell lines moderately increased ß-catenin mediated transcription, which contrasts with previous data showing SOX17 is an inhibitor of TCF/ß-catenin signaling. The proliferation of EC cell lines was expectedly reduced by transfection with SOX17-WT, and further reduced by SOX17-A96G and SOX17-S403I. These data implicate SOX17 mutation as a selected event in EEC, with clear differences between the missense and frameshift mutations.

Subtype-Specific Tumor-Associated Fibroblasts Contribute to the Pathogenesis of Uterine Leiomyoma.

Recent genomic studies have identified subtypes of uterine leiomyoma (LM) with distinctive genetic alterations. Here, we report the elucidation of the biological characteristics of the two most prevalent uterine leiomyoma subtypes, MED12-mutant (MED12-LM) and HMGA2-overexpressing (HMGA2-LM) uterine leiomyomas. Because each tumor carries only one genetic alteration, both subtypes are considered to be monoclonal. Approximately 90% of cells in HMGA2-uterine leiomyoma were smooth muscle cells (SMC) with HMGA2 overexpression. In contrast, MED12-LM consisted of similar numbers of SMC and non-SMC, which were mostly tumor-associated fibroblasts (TAF). Paradoxically, TAF carried no mutations in MED12, suggesting an interaction between SMC and TAF to coordinate their growth. The higher amount of extracellular matrix in MED12-LM than HMGA2-LM was partially due to the high concentration of collagen-producing TAF. SMC growth in a xenograft assay was driven by progesterone in both uterine leiomyoma subtypes. In contrast, TAF in MED12-LM proliferated in response to estradiol, whereas progesterone had no effect. The high concentration of estrogen-responsive TAF in MED12-LM explains the inconsistent discoveries between in vivo and in vitro studies on the mitogenic effect of estrogen and raises questions regarding the accuracy of previous studies utilizing MED12-LM cell culture. In addition, the differential effects of estradiol and progesterone on these uterine leiomyoma subtypes emphasize the importance of subtypes and genotypes in designing nonsurgical therapeutic strategies for uterine leiomyoma. Cancer Res; 77(24); 6891-901. ©2017 AACR.

Halofuginone suppresses growth of human uterine leiomyoma cells in a mouse xenograft model.

STUDY QUESTION: Does halofuginone (HF) inhibit the growth of human uterine leiomyoma cells in a mouse xenograft model? SUMMARY ANSWER: HF suppresses the growth of human uterine leiomyoma cells in a mouse xenograft model through inhibiting cell proliferation and inducing apoptosis. WHAT IS KNOWN ALREADY: Uterine leiomyomas are the most common benign tumors of the female reproductive tract. HF can suppress the growth of human uterine leiomyoma cells in vitro. The mouse xenograft model reflects the characteristics of human leiomyomas. STUDY DESIGN, SIZE, DURATION: Primary leiomyoma smooth muscle cells from eight patients were xenografted under the renal capsule of adult, ovariectomized NOD-scid IL2Rγ(null) mice (NSG). Mice were treated with two different doses of HF or vehicle for 4 weeks with six to eight mice per group. PARTICIPANTS/MATERIALS, SETTING, METHODS: Mouse body weight measurements and immunohistochemical analysis of body organs were carried out to assess the safety of HF treatment. Xenografted tumors were measured and analyzed for cellular and molecular changes induced by HF. Ovarian steroid hormone receptors were evaluated for possible modulation by HF. MAIN RESULTS AND THE ROLE OF CHANCE: Treatment of mice carrying human UL xenografts with HF at 0.25 or 0.50 mg/kg body weight for 4 weeks resulted in a 35-40% (P < 0.05) reduction in tumor volume. The HF-induced volume reduction was accompanied by increased apoptosis and decreased cell proliferation. In contrast, there was no significant change in the collagen content either at the transcript or protein level between UL xenografts in control and HF groups. HF treatment did not change the expression level of ovarian steroid hormone receptors. No adverse pathological effects were observed in other tissues from mice undergoing treatment at these doses. LIMITATIONS, REASONS FOR CAUTION: While this study did test the effects of HF on human leiomyoma cells in an in vivo model, HF was administered to mice whose tolerance and metabolism of the drug may differ from that in humans. Also, the longer term effects of HF treatment are yet unclear. WIDER IMPLICATIONS OF THE FINDINGS: The results of this study showing the effectiveness of HF in reducing UL tumor growth by interfering with the main cellular processes regulating cell proliferation and apoptosis are in agreement with previous studies on the effects of HF on other fibrotic diseases. HF can be considered as a candidate for reducing the size of leiomyomas, particularly prior to surgery. STUDY FUNDING/COMPETING INTERESTS: This project was funded by NIH PO1HD057877 and R01 HD064402. Authors report no competing interests.

FGFR2IIIb-MAPK Activity Is Required for Epithelial Cell Fate Decision in the Lower Müllerian Duct.

Cell fate of lower Müllerian duct epithelium (MDE), to become uterine or vaginal epithelium, is determined by the absence or presence of ΔNp63 expression, respectively. Previously, we showed that SMAD4 and runt-related transcription factor 1 (RUNX1) were independently required for MDE to express ΔNp63. Here, we report that vaginal mesenchyme directs vaginal epithelial cell fate in MDE through paracrine activation of fibroblast growth factor (FGF) receptor-MAPK pathway. In the developing reproductive tract, FGF7 and FGF10 were enriched in vaginal mesenchyme, whereas FGF receptor 2IIIb was expressed in epithelia of both the uterus and vagina. When Fgfr2 was inactivated, vaginal MDE underwent uterine cell fate, and this differentiation defect was corrected by activation of MEK-ERK pathway. In vitro, FGF10 in combination with bone morphogenetic protein 4 and activin A (ActA) was sufficient to induce ΔNp63 in MDE, and ActA was essential for induction of RUNX1 through SMAD-independent pathways. Accordingly, inhibition of type 1 receptors for activin in neonatal mice induced uterine differentiation in vaginal epithelium by down-regulating RUNX1, whereas conditional deletion of Smad2 and Smad3 had no effect on vaginal epithelial differentiation. In conclusion, vaginal epithelial cell fate in MDE is induced by FGF7/10-MAPK, bone morphogenetic protein 4-SMAD, and ActA-RUNX1 pathway activities, and the disruption in any one of these pathways results in conversion from vaginal to uterine epithelial cell fate.

Human uterine leiomyoma stem/progenitor cells expressing CD34 and CD49b initiate tumors in vivo.

CONTEXT: Uterine leiomyoma is the most common benign tumor in reproductive-age women. Using a dye-exclusion technique, we previously identified a side population of leiomyoma cells exhibiting stem cell characteristics. However, unless mixed with mature myometrial cells, these leiomyoma side population cells did not survive or grow well in vitro or in vivo. OBJECTIVE: The objective of this study was to identify cell surface markers to isolate leiomyoma stem/progenitor cells. DESIGN: Real-time PCR screening was used to identify cell surface markers preferentially expressed in leiomyoma side population cells. In vitro colony-formation assay and in vivo tumor-regeneration assay were used to demonstrate functions of leiomyoma stem/progenitor cells. RESULTS: We found significantly elevated CD49b and CD34 gene expression in side population cells compared with main population cells. Leiomyoma cells were sorted into three populations based on the expression of CD34 and CD49b: CD34(+)/CD49b(+), CD34(+)/CD49b(-), and CD34(-)/CD49b(-) cells, with the majority of the side population cells residing in the CD34(+)/CD49b(+) fraction. Of these populations, CD34(+)/CD49b(+) cells expressed the lowest levels of estrogen receptor-α, progesterone receptor, and α-smooth muscle actin, but the highest levels of KLF4, NANOG, SOX2, and OCT4, confirming their more undifferentiated status. The stemness of CD34(+)/CD49b(+) cells was also demonstrated by their strongest in vitro colony-formation capacity and in vivo tumor-regeneration ability. CONCLUSIONS: CD34 and CD49b are cell surface markers that can be used to enrich a subpopulation of leiomyoma cells possessing stem/progenitor cell properties; this technique will accelerate efforts to develop new therapies for uterine leiomyoma.

Diethylstilbestrol induces vaginal adenosis by disrupting SMAD/RUNX1-mediated cell fate decision in the Müllerian duct epithelium.

Women exposed to diethylstilbestrol (DES) in utero frequently develop vaginal adenosis, from which clear cell adenocarcinoma can arise. Despite decades of extensive investigation, the molecular pathogenesis of DES-associated vaginal adenosis remains elusive. Here we report that DES induces vaginal adenosis by inhibiting the BMP4/Activin A-regulated vaginal cell fate decision through a downregulation of RUNX1. BMP4 and Activin A produced by vaginal mesenchyme synergistically activated the expression of ΔNp63, thus deciding vaginal epithelial cell fate in the Müllerian duct epithelial cells (MDECs) via direct binding of SMADs on the highly conserved 5' sequence of ΔNp63. Therefore, mice in which Smad4 was deleted in MDECs failed to express ΔNp63 in vaginal epithelium and developed adenosis. This SMAD-dependent ΔNp63 activation required RUNX1, a binding partner of SMADs. Conditional deletion of Runx1 in the MDECs induced adenosis in the cranial portion of vagina, which mimicked the effect of developmental DES-exposure. Furthermore, neonatal DES exposure downregulated RUNX1 in the fornix of the vagina, where DES-associated adenosis is frequently found. This observation strongly suggests that the downregulation of RUNX1 is the cause of vaginal adenosis. However, once cell fate was determined, the BMP/Activin-SMAD/RUNX1 signaling pathway became dispensable for the maintenance of ΔNp63 expression in vaginal epithelium. Instead, the activity of the ΔNp63 locus in vaginal epithelium was maintained by a ΔNp63-dependent mechanism. This is the first demonstration of a molecular mechanism through which developmental chemical exposure causes precancerous lesions by altering cell fate.

Hydroxyitraconazole, formed during intestinal first-pass metabolism of itraconazole, controls the time course of hepatic CYP3A inhibition and the bioavailability of itraconazole in rats.

Itraconazole (ITZ) is a substrate of CYP3A and both ITZ and hydroxyitraconazole (OH-ITZ), a major metabolite formed by CYP3A, are potent inhibitors of CYP3A. The concentration- and time-dependent changes in the hepatic availability (F(H)) of ITZ were evaluated in rats after oral doses of 5 and 40 mg/kg. Simultaneous blood samples were obtained from the aorta, portal vein, and hepatic vein for 24 h following duodenal ITZ administration, and concentrations of ITZ and OH-ITZ determined by LC/MS. During the absorption phase, the F(H) of ITZ increased from 0.2 to 1.0, reflecting the time course of hepatic CYP3A inhibition. A counterclockwise hysteresis was observed between ITZ concentrations entering the liver (C(IN,ITZ)) and F(H), whereas there was no time delay observed between the change in F(H) and the OH-ITZ concentrations entering the liver (C(IN,OH-ITZ)). The direct relationship between C(IN,OH-ITZ) and F(H) suggested that OH-ITZ was mainly responsible for the inhibition of CYP3A. A positive portal venous-aortic gradient for OH-ITZ was measured after duodenal administration of ITZ, indicating intestinal formation of OH-ITZ. The in vivo Ki for OH-ITZ (38 +/- 3 nM) was estimated from C(IN,OH-ITZ) versus F(H) of ITZ, and is similar to values obtained from inhibition of midazolam hydroxylation in CYP3A4 supersomes (Drug Metab Dispos 32:1121-1131, 2004). The data suggest that OH-ITZ, formed by intestinal CYP3A, controls the time course of hepatic CYP3A inhibition and is mainly responsible for the observed increase in F(H) of ITZ.

The effect of anesthesia and surgery on CYP3A activity in rats.

The purpose of this investigation was to examine the effects of surgery and anesthesia on in vivo CYP3A activity and portal venous blood flow. Midazolam, a CYP3A probe for both rats and humans, was administered orally (2.7 mg), intravenously (0.57 mg), or via the portal vein (0.57 mg) to rats 4 h after anesthesia with ketamine/xylazine and surgery for placement of indwelling vascular and duodenal catheters and 3 days after surgery (chronic). The systemic clearance of midazolam was 51 +/- 4 ml/min/kg in the chronic animals, and this was significantly decreased (29 +/- 1 ml/min/kg, P = 0.024) in acute rats studied 4 to 6 h after anesthesia and surgery. The hepatic availability (FH), directly determined from the aortic and hepatic venous concentration gradient, was significantly higher in the acute animals (0.57 +/- 0.05) compared with the chronic animals (0.33 +/- 0.07, P = 0.001). Hepatic availability was determined using a classical approach in which FH was calculated from the area under the plasma concentration versus time curve ratio after portal venous or intravenous administration. FH was higher in the acute rats (0.48) compared with the chronic animals (0.27 +/- 0.03). Portal venous blood flow was significantly lower in the acute animals (5.0 +/- 0.4 ml/min/100 g body weight) compared with the chronic animals (9.1 +/- 0.9 ml/min/100 g body weight, P = 0.015). The effect of surgery and anesthesia was confirmed using the indicator dye dilution method after infusion of [14C]polyethylene glycol 4000 into the superior mesenteric artery. Our data suggest that anesthesia and surgery decreases both hepatic CYP3A activity and hepatic blood flow in rats. Studies performed in rats within 3 days of surgery and anesthesia are conducted under nonphysiologic conditions and therefore provide inaccurate assessment of drug disposition, in particular, clearance and bioavailability.

Differential induction of hepatic dysfunction after intraportal and intravenous challenge with endotoxin and Staphylococcal enterotoxin B.

We have previously shown that systemic infusion of the bacterial toxins Staphylococcal enterotoxin B (SEB) and endotoxin (LPS) induces hepatic dysfunction as measured by decreased biliary indocyanine green (ICG) excretion. In this study, we compare the effects of these bacterial toxins after infusion into the portal and systemic circulation and directly measure biliary bile acid excretion as a measure of cholestasis. We hypothesized that bacterial toxins infused into the portal vein would induce greater hepatic dysfunction than toxins infused into the systemic circulation. Using a chronically catheterized rat model, biliary bile acid excretion was directly measured after infusion of LPS at 10 and 100 microg/kg with and without 50 microg/kg SEB into the portal vein (IPV) or inferior vena cava (IV) at baseline, and at 6 and 24 h. We found that when LPS was infused alone, only IPV administration caused a significant decrease in bile acid excretion at 6 h. There was no change in bile acid excretion after IV administration of LPS. In contrast, when the combination of LPS and SEB was infused, both IV and IPV administration significantly decreased bile acid excretion at 6 and 24 h. At 6 h post-LPS and -SEB administration, the decrease in bile acid excretion was significantly greater after IPV than IV administration. There was no site-specific difference in IFN-gamma release after infusion of toxins. However, peak TNFalpha release was decreased in IPV-infused rats [10 microg/kg (P < 0.05) or 100 microg/kg (P = ns) LPS with SEB] compared with the same doses in IV-infused rats. These data question the role of systemic TNF-alpha and IFN-gamma in regulating hepatic dysfunction and suggest a differential functional response of the liver to systemic and gut-derived septic events. This study also further explains the frequent development of liver dysfunction in patients with sepsis, multisystem organ failure, and other diseases with altered intestinal permeability.

Chronic Staphylococcal enterotoxin B and lipopolysaccharide induce a bimodal pattern of hepatic dysfunction and injury.

OBJECTIVE: To determine the effect of chronic exposure to endotoxin (lipopolysaccharide) and Staphylococcal enterotoxin B on hepatic injury and function. DESIGN: Prospective, controlled trial. SETTING: Research laboratory in a university hospital. SUBJECTS: Male Sprague-Dawley rats weighing 325-350 g with chronic vascular and bile catheters. INTERVENTIONS: Chronically catheterized rats were treated daily with saline, 50 microg/kg Staphylococcal enterotoxin B alone, 1000 microg/kg lipopolysaccharide alone, 1000 microg/kg lipopolysaccharide with 50 microg/kg Staphylococcal enterotoxin B, or 100 microg/kg lipopolysaccharide with 50 microg/kg Staphylococcal enterotoxin B for 10 days. Serum and biliary measures of hepatic injury and dysfunction were measured before and then 6 hrs and 1, 2, 3, 7, and 10 days after the start of treatment. The animals were killed at 10 days and the livers examined histologically. MEASUREMENTS AND MAIN RESULTS: Mean rates of bile flow, biliary indocyanine green excretion, and bile acid flux were significantly decreased immediately after treatment (6 hr, 1 and 2 days) and then at 10 days. Increases in biliary and serum gamma-glutamyltransferase and serum bile acids also occurred in a similar bimodal pattern. Animals treated with lipopolysaccharide or Staphylococcal enterotoxin B alone became tolerant and did not develop the bimodal pattern of hepatic dysfunction. Histologic examination of the liver at 10 days revealed periportal inflammation and fibrosis. CONCLUSIONS: The combination of lipopolysaccharide and Staphylococcal enterotoxin B leads to late liver injury, whereas either toxin alone does not. These data may explain the frequent development of liver dysfunction in patients exposed to multiple bacterial toxins such as in sepsis, multiple-system organ failure, and other diseases with altered intestinal permeability.

Splenectomy ablates endotoxin-induced IFNgamma response in rats.

The mechanism of liver injury in endotoxemia is unclear. Previous studies have shown that splenectomy protects the liver from endotoxin-induced injury. The purpose of this study was to determine the relationship of TNFalpha and IFNgamma release and endotoxin-induced liver injury in splenectomized and nonsplenectomized rats. Splenectomized and nonsplenectomized (Sham) rats with chronic catheters in the aorta and inferior vena cava (IVC) were parenterally infused with 10 to 5000 microg/kg endotoxin. TNFalpha, IFNgamma, and alanine aminotransferase (ALT), a marker of hepatocellular damage, were measured in aortic blood. Compared to sham controls, splenectomized animals demonstrated significantly reduced endotoxin-induced ALT concentrations at endotoxin doses >10 microg/kg. Peak endotoxin-induced TNFalpha concentrations were not significantly different between the splenectomized and sham groups. In contrast, peak endotoxin-induced IFNgamma concentrations were significantly decreased in the splenectomized group. These data suggest a relationship between endotoxin-induced IFNgamma and liver injury. We speculate that the spleen contributes to the endotoxin-induced liver injury by modulating release of IFNgamma.