Niclosamide's potential direct targets in ovarian cancer†.

Recent evidence indicates that niclosamide is an anti-cancer compound that is able to inhibit several signaling pathways. Although niclosamide has previously been identified by high-throughput screening platforms as a potential effective compound against several cancer types, no direct binding interactions with distinct biological molecule(s) has been established. The present study identifies key signal transduction mechanisms altered by niclosamide in ovarian cancer. Using affinity purification with a biotin-modified niclosamide derivative and mass spectrometry analysis, several RNA-binding proteins (RBPs) were identified. We chose the two RBPs, FXR1 and IGF2BP2, for further analysis. A significant correlation exists in which high-expression of FXR1 or IGF2BP2 is associated with reduced survival of ovarian cancer patients. Knockdown of FXR1 or IGF2BP2 in ovarian cancer cells resulted in significantly reduced cell viability, adhesion, and migration. Furthermore, FXR1 or IGF2BP2 deficient ovarian cancer cells exhibited reduced response to most doses of niclosamide showing greater cell viability than those with intact RBPs. These results suggest that FXR1 and IGF2BP2 are direct targets of niclosamide and could have critical activities that drive multiple oncogenic pathways in ovarian cancer.

AMPK is required for uterine receptivity and normal responses to steroid hormones.

We previously demonstrated that 5'-AMP-activated protein kinase (AMPK) is essential for normal reproductive functions in female mice. Conditional ablation of Prkaa1 and Prkaa2, genes that encode the α1 and α2 catalytic domains of AMPK, resulted in early reproductive senescence, faulty artificial decidualization, uterine inflammation and fibrotic postparturient endometrial regeneration. We also noted a delay in the timing of embryo implantation in Prkaa1/2d/d female mice, suggesting a role for AMPK in establishing uterine receptivity. As outlined in new studies here, conditional uterine ablation of Prkaa1/2 led to an increase in ESR1 in the uteri of Prkaa1/2d/d mice, resulting in prolonged epithelial cell proliferation and retention of E2-induced gene expression (e.g. Msx1, Muc1, Ltf) through the implantation window. Within the stromal compartment, stromal cell proliferation was reduced by five-fold in Prkaa1/2d/d mice, and this was accompanied by a significant decrease in cell cycle regulatory genes and aberrant expression of decidualization marker genes such as Hand2, Bmp2, Fst and Inhbb. This phenotype is consistent with our prior study, demonstrating a failure of the Prkaa1/2d/d uterus to undergo decidualization. Despite these uterine defects, ovarian function seemed to be normal following ablation of Prkaa1/2 from peri-ovulatory follicles in which ovulation, luteinization and serum progesterone levels were not different on day 5 of pregnancy or pseudopregnancy between Prkaa1/2fl/fl and Prkaa1/2d/d mice. These cumulative findings demonstrate that AMPK activity plays a prominent role in mediating several steroid hormone-dependent events such as epithelial cell proliferation, uterine receptivity and decidualization as pregnancy is established.

Forkhead box a2 (FOXA2) is essential for uterine function and fertility.

Establishment of pregnancy is a critical event, and failure of embryo implantation and stromal decidualization in the uterus contribute to significant numbers of pregnancy losses in women. Glands of the uterus are essential for establishment of pregnancy in mice and likely in humans. Forkhead box a2 (FOXA2) is a transcription factor expressed specifically in the glands of the uterus and is a critical regulator of postnatal uterine gland differentiation in mice. In this study, we conditionally deleted FOXA2 in the adult mouse uterus using the lactotransferrin Cre (Ltf-Cre) model and in the neonatal mouse uterus using the progesterone receptor Cre (Pgr-Cre) model. The uteri of adult FOXA2-deleted mice were morphologically normal and contained glands, whereas the uteri of neonatal FOXA2-deleted mice were completely aglandular. Notably, adult FOXA2-deleted mice are completely infertile because of defects in blastocyst implantation and stromal cell decidualization. Leukemia inhibitory factor (LIF), a critical implantation factor of uterine gland origin, was not expressed during early pregnancy in adult FOXA2-deleted mice. Intriguingly, i.p. injections of LIF initiated blastocyst implantation in the uteri of both gland-containing and glandless adult FOXA2-deleted mice. Although pregnancy was rescued by LIF and was maintained to term in uterine gland-containing adult FOXA2-deleted mice, pregnancy failed by day 10 in neonatal FOXA2-deleted mice lacking uterine glands. These studies reveal a previously unrecognized role for FOXA2 in regulation of adult uterine function and fertility and provide original evidence that uterine glands and, by inference, their secretions play important roles in blastocyst implantation and stromal cell decidualization.

Progesterone receptor membrane component 1 and 2 regulate granulosa cell mitosis and survival through a NFΚB-dependent mechanism†.

Progesterone receptor membrane component 1 (PGRMC1) interacts with PGRMC2, and disrupting this interaction in spontaneously immortalized granulosa cells (SIGCS) leads to an inappropriate entry into the cell cycle, mitotic arrest, and ultimately cell death. The present study revealed that PGRMC1 and PGRMC2 localize to the cytoplasm of murine granulosa cells of nonatretric follicles with their staining intensity being somewhat diminished in granulosa cells of atretic follicles. Compared to controls (Pgrmc1fl/fl), the rate at which granulosa cells entered the cell cycle increased in nonatretic and atretic follicles of mice in which Pgrmc1 was conditionally deleted (Pgrmc1d/d) from granulosa cells. This increased rate of entry into the cell cycle was associated with a ≥ 2-fold increase in follicular atresia and the nuclear localization of nuclear factor-kappa-B transcription factor P65; (NFΚB/p65, or RELA). GTPase activating protein binding protein 2 (G3BP2) binds NFΚB/p65 through an interaction with NFΚB inhibitor alpha (IκBα), thereby maintaining NFΚB/p65's cytoplasmic localization and restricting its transcriptional activity. Since PGRMC1 and PGRMC2 bind G3BP2, studies were designed to assess the functional relationship between PGRMC1, PGRMC2, and NFΚB/p65 in SIGCs. In these studies, disrupting the interaction between PGRMC1 and PGRMC2 increased the nuclear localization of NFΚB/p65, and depleting PGRMC1, PGRMC2, or G3BP2 increased NFΚB transcriptional activity and the progression into the cell cycle. Taken together, these studies suggest that PGRMC1 and 2 regulate granulosa cell cycle entry in follicles by precisely controlling the localization and thereby the transcriptional activity of NFΚB/p65.

PGRMC1/2 promotes luteal vascularization and maintains the primordial follicles of mice

To determine whether conditional depletion of progesterone receptor membrane component (PGRMC) 1 and PGRMC2 affected ovarian follicle development, follicle distribution was assessed in ovaries of young (≈3-month-old) and middle-aged (≈6-month-old) control (Pgrmc1/2fl/fl) and double conditional PGRMC1/2-knockout (Pgrmc1/2d/d) mice. This study revealed that the distribution of primary, preantral and antral follicles was not altered in Pgrmc1/2d/d mice, regardless of the age. Although the number of primordial follicles was similar at ≈3 months of age, their numbers were reduced by ≈80% in 6-month-old Pgrmc1/2d/d mice compared to age-matched Pgrmc1/2fl/fl mice. The Pgrmc1/2d/d mice were generated using Pgr-cre mice, so ablation of Pgrmc1 and Pgrmc2 in the ovary was restricted to peri-ovulatory follicles and subsequent corpora lutea (CL). In addition, the vascularization of CL was attenuated in Pgrmc1/2d/d mice, although mRNA levels of vascular endothelial growth factor A (Vegfa) were elevated. Moreover, depletion of Pgrmc1 and Pgrmc2 altered the gene expression profile in the non-luteal component of the ovary such that Vegfa expression, a stimulator of primordial follicle growth, was elevated; Kit Ligand expression, another stimulator of primordial follicle growth, was suppressed and anti-Mullerian hormone, an inhibitor of primordial follicle growth, was enhanced compared to Pgrmc1/2fl/fl mice. These data reveal that luteal cell depletion of Pgrmc1 and 2 alters the expression of growth factors within the non-luteal component of the ovary, which could account for the premature demise of the adult population of primordial follicles. In summary, the survival of adult primordial follicles is dependent in part on progesterone receptor membrane component 1 and 2.

A functional role for AMPK in female fertility and endometrial regeneration.

Adenosine monophosphate-activated protein kinase (AMPK) is a highly conserved heterotrimeric complex that acts as an intracellular energy sensor. Based on recent observations of AMPK expression in all structures of the female reproductive system, we hypothesized that AMPK is functionally required for maintaining fertility in the female. This hypothesis was tested by conditionally ablating the two catalytic alpha subunits of AMPK, Prkaa1 and Prkaa2, using Pgr-cre mice. After confirming the presence of PRKAA1, PRKAA2 and the active phospho-PRKAA1/2 in the gravid uterus by immunohistochemistry, control (Prkaa1/2 fl/fl ) and double conditional knockout mice (Prkaa1/2 d/d ) were placed into a six-month breeding trial. While the first litter size was comparable between Prkaa1/2 fl/fl and Prkaa1/2 d/d female mice (P = 0.8619), the size of all subsequent litters was dramatically reduced in Prkaa1/2 d/d female mice (P = 0.0015). All Prkaa1/2 d/d female mice experienced premature reproductive senescence or dystocia by the fourth parity. This phenotype manifested despite no difference in estrous cycle length, ovarian histology in young and old nulliparous or multiparous animals, mid-gestation serum progesterone levels or uterine expression of Esr1 or Pgr between Prkaa1/2 fl/fl and Prkaa1/2 d/d female mice suggesting that the hypothalamic-pituitary-ovary axis remained unaffected by PRKAA1/2 deficiency. However, an evaluation of uterine histology from multiparous animals identified extensive endometrial fibrosis and disorganized stromal-glandular architecture indicative of endometritis, a condition that causes subfertility or infertility in most mammals. Interestingly, Prkaa1/2 d/d female mice failed to undergo artificial decidualization. Collectively, these findings suggest that AMPK plays an essential role in endometrial regeneration following parturition and tissue remodeling that accompanies decidualization.

Expression of progesterone receptor membrane component-2 within the immature rat ovary and its role in regulating mitosis and apoptosis of spontaneously immortalized granulosa cells.

Progesterone receptor membrane component 2 (Pgrmc2) mRNA was detected in the immature rat ovary. By 48 h after eCG, Pgrmc2 mRNA levels decreased by 40% and were maintained at 48 h post-hCG. Immunohistochemical studies detected PGRMC2 in oocytes and ovarian surface epithelial, interstitial, thecal, granulosa, and luteal cells. PGRMC2 was also present in spontaneously immortalized granulosa cells, localizing to the cytoplasm of interphase cells and apparently to the mitotic spindle of cells in metaphase. Interestingly, PGRMC2 levels appeared to decrease during the G1 stage of the cell cycle. Moreover, overexpression of PGRMC2 suppressed entry into the cell cycle, possibly by binding the p58 form of cyclin dependent kinase 11b. Conversely, Pgrmc2 small interfering RNA (siRNA) treatment increased the percentage of cells in G1 and M stage but did not increase the number of cells, which was likely due to an increase in apoptosis. Depleting PGRMC2 did not inhibit cellular (3)H-progesterone binding, but attenuated the ability of progesterone to suppress mitosis and apoptosis. Taken together these studies suggest that PGRMC2 affects granulosa cell mitosis by acting at two specific stages of the cell cycle. First, PGRMC2 regulates the progression from the G0 into the G1 stage of the cell cycle. Second, PGRMC2 appears to localize to the mitotic spindle, where it likely promotes the final stages of mitosis. Finally, siRNA knockdown studies indicate that PGRMC2 is required for progesterone to slow the rate of granulosa cell mitosis and apoptosis. These findings support a role for PGRMC2 in ovarian follicle development.

Nonerythroid hemoglobin promotes human cumulus cell viability and the developmental capacity of the human oocyte.

OBJECTIVE: To determine the relationship between the levels of cumulus cell (CC) hemoglobin messenger ribonucleic acid (mRNA) and the developmental potential of the associated oocyte and whether hemoglobin protects the CCs from oxidative stress-induced apoptosis. DESIGN: Laboratory-based study. SETTING: University laboratory and university-affiliated in vitro fertilization center. PATIENT(S): Cumulus cells from the oocytes of patients who underwent in vitro fertilization with intracytoplasmic sperm injection with and without preimplantation genetic testing between 2018 and 2020. INTERVENTION(S): Studies on individual and pooled CCs collected at the time of oocyte retrieval or cultured under 20% or 5% O2. MAIN OUTCOME MEASURE(S): Quantitative polymerase chain reaction analysis of individual and pooled patient CC samples were used to monitor the hemoglobin mRNA levels. Reverse transcription-polymerase chain reaction arrays were used to assess genes that regulate oxidative stress in CCs associated with aneuploid and euploid blastocysts. Studies were conducted to assess the effect of oxidative stress on the rate of apoptosis, level of reactive oxygen species, and gene expression in CCs in vitro. RESULT(S): Compared with CCs associated with arrested and aneuploid blastocysts, the mRNA levels encoding the alpha and beta chains of hemoglobin increased by 2.9- and 2.3-fold in CCs associated with euploid blastocysts, respectively. The mRNA levels encoding the alpha and beta chains of hemoglobin also increased by 3.8- and 4.5-fold in CCs cultured under 5% O2 vs. 20% O2, respectively, and multiple regulators of oxidative stress were overexpressed in cells cultured under 20% O2 compared with those under 5% O2. However, the rate of apoptosis and amount of mitochondrial reactive oxidative species increased by 1.25-fold in CCs cultured under 20% O2 compared with those under 5% O2. Variable amounts of the alpha and beta chains of hemoglobin were also detected within the zona pellucida and oocytes. CONCLUSION(S): Higher levels of nonerythroid hemoglobin in CCs are associated with oocytes that result in euploid blastocysts. Hemoglobin may protect CCs from oxidative stress-induced apoptosis, which may enhance cumulus-oocyte interactions. Moreover, CC-derived hemoglobin may be transferred to the oocytes and protect it from the adverse effects of oxidative stress that occurs in vivo and in vitro.

STAT3 Knockdown Induces Tumor Formation by MDA-MB-231 Cells.

STAT3 plays a central role in oncogenesis by mediating cell survival, growth, and differentiation. It is constitutively activated in breast cancer. We investigated the role of STAT3 in tumor development by knocking down STAT3 levels in MDA-MB-231 triple negative breast cancer cells using short hairpin RNA. The tumor forming potential of these STAT3-depleted cells was assessed by xenografts in immunocompromised NOD SCID mice. Contrary to its accepted tumor promoting role, we found STAT3 to be a negative regulator of growth in MDA-MB-231- derived tumors. Although similar observations have been made in thyroid carcinoma and lung adenocarcinoma xenograft studies, our novel results showed for the first time that the role of STAT3 in promoting tumorigenesis may be context-specific, and that STAT3 may actually be a negative regulator of certain breast-cancer types. Studies to identify the mechanisms of STAT3's negative regulatory role may be useful in developing STAT3-based therapeutics.

Conditional Ablation of Progesterone Receptor Membrane Component 2 Causes Female Premature Reproductive Senescence.

The nonclassical progesterone receptors progesterone receptor membrane component (PGRMC) 1 and PGRMC2 have been implicated in regulating cell survival of endometrial and ovarian cells in vitro and are abundantly expressed in these cell types. The objective of this study was to determine if Pgrmc1 and Pgrmc2 are essential for normal female reproduction. To accomplish this objective, Pgrmc1 and/or Pgrmc2 floxed mice (Pgrmc2fl/fl and Pgrmc1/2fl/fl) were crossed with Pgr-cre mice, which resulted in the conditional ablation of Pgrmc1 and/or Pgrmc2 from female reproductive tissues (i.e.,Pgrmc2d/d and Pgrmc1/2d/d mice). A breeding trial revealed that conditional ablation of Pgrmc2 initially led to subfertility, with Pgrmc2d/d female mice producing 47% fewer pups/litter than Pgrmc2fl/fl mice (P = 0.001). Pgrmc2d/d mice subsequently underwent premature reproductive senescence by parities 2 to 5, producing 37.8% fewer litters overall during the trial compared with Pgrmc2fl/fl mice (P = 0.020). Similar results were observed with Pgrmc1/2d/d mice. Based on ovarian morphology and serum P4, the subfertility/infertility was not due to faulty ovulation or luteal insufficiency. Rather an analysis of midgestation implantation sites revealed that postimplantation embryonic death was the major cause of the subfertility/infertility. As with our previous report of Pgrmc1d/d mice, Pgrmc2d/d and Pgrmc1/2d/d mice developed endometrial cysts consistent with accelerated aging of this tissue. Given the timing of postimplantation embryonic demise, uterine decidualization may be disrupted in mice deficient in PGRMC2 or PGRMC1/2. Overall, this study revealed that Pgrmc1 and/or Pgrmc2 are required for the maintenance of uterine histoarchitecture and normal female reproductive lifespan.

Progesterone receptor membrane component 1 promotes survival of human breast cancer cells and the growth of xenograft tumors.

Triple negative breast cancers (TNBCs) are highly aggressive and grow in response to sex steroid hormones despite lacking expression of the classical estrogen (E2) and progesterone (P4) receptors. Since P4 receptor membrane component 1 (PGRMC1) is expressed in breast cancer tumors and is known to mediate P4-induced cell survival, this study was designed to determine the expression of PGRMC1 in TNBC tumors and the involvement of PGRMC1 in regulating proliferation and survival of TNBC cells in vitro and the growth of TNBC tumors in vivo. For the latter studies, the MDA-MB-231 (MDA) cell line derived from TNBC was used. These cells express PGRMC1 but lack expression of the classical P4 receptor. A lentiviral-based shRNA approach was used to generate a stably transfected PGRMC1-deplete MDA line for comparison to the PGRMC1-intact MDA line. The present studies demonstrate that PGRMC1: 1) is expressed in TNBC cells; 2) mediates the ability of P4 to suppress TNBC cell mitosis in vitro; 3) is required for P4 to reduce the apoptotic effects of doxorubicin in vitro; and 4) facilitates TNBC tumor formation and growth in vivo. Taken together, these findings indicate that PGRMC1 plays an important role in regulating the growth and survival of TNBC cells in vitro and ultimately in the formation and development of these tumors in vivo. Thus, PGRMC1 may be a therapeutic target for TNBCs.

Conditional Ablation of Progesterone Receptor Membrane Component 1 Results in Subfertility in the Female and Development of Endometrial Cysts.

Progesterone (P4) is essential for female fertility. The objective of this study was to evaluate the functional requirement of the nonclassical P4 receptor (PGR), PGR membrane component 1, in regulating female fertility. To achieve this goal, the Pgrmc1 gene was floxed by insertion of loxP sites on each side of exon 2. Pgrmc1 floxed (Pgrmc1(fl/fl)) mice were crossed with Pgr(cre) or Amhr2(cre) mice to delete Pgrmc1 (Pgrmc1(d/d)) from the female reproductive tract. A 6-month breeding trial revealed that conditional ablation of Pgrmc1 with Pgr(cre/+) mice resulted in a 40% reduction (P = .0002) in the number of pups/litter. Neither the capacity to ovulate in response to gonadotropin treatment nor the expression of PGR and the estrogen receptor was altered in the uteri of Pgrmc1(d/d) mice compared with Pgrmc1(fl/fl) control mice. Although conditional ablation of Pgrmc1 from mesenchymal tissue using Amhr2(cre/+) mice did not reduce the number of pups/litter, the total number of litters born in the 6-month breeding trial was significantly decreased (P = .041). In addition to subfertility, conditional ablation of Pgrmc1 using either Amhr2(cre/+) or Pgr(cre/+) mice resulted in the development of endometrial cysts starting around 4 months of age. Interestingly, pregnancy attenuated the formation of these uterine cysts. These new findings demonstrate that PGR membrane component 1 plays an important role in female fertility and uterine tissue homeostasis.

Progesterone receptor membrane component 1 deficiency attenuates growth while promoting chemosensitivity of human endometrial xenograft tumors.

Endometrial cancer is the leading gynecologic cancer in women in the United States with 52,630 women predicted to be diagnosed with the disease in 2014. The objective of this study was to determine if progesterone (P4) receptor membrane component 1 (PGRMC1) influenced endometrial cancer cell viability in response to chemotherapy in vitro and in vivo. A lentiviral-based shRNA knockdown approach was used to generate stable PGRMC1-intact and PGRMC1-deplete Ishikawa endometrial cancer cell lines that also lacked expression of the classical progesterone receptor (PGR). Progesterone treatment inhibited mitosis of PGRMC1-intact, but not PGRMC1-deplete cells, suggesting that PGRMC1 mediates the anti-mitotic actions of P4. To test the hypothesis that PGRMC1 attenuates chemotherapy-induced apoptosis, PGRMC1-intact and PGRMC1-deplete cells were treated in vitro with vehicle, P4 (1 µM), doxorubicin (Dox, 2 µg/ml), or P4 + Dox for 48 h. Doxorubicin treatment of PGRMC1-intact cells resulted in a significant increase in cell death; however, co-treatment with P4 significantly attenuated Dox-induced cell death. This response to P4 was lost in PGRMC1-deplete cells. To extend these observations in vivo, a xenograft model was employed where PGRMC1-intact and PGRMC1-deplete endometrial tumors were generated following subcutaneous and intraperitoneal inoculation of immunocompromised NOD/SCID and nude mice, respectively. Tumors derived from PGRMC1-deplete cells grew slower than tumors from PGRMC1-intact cells. Mice harboring endometrial tumors were then given three treatments of vehicle (1:1 cremophor EL: ethanol + 0.9% saline) or chemotherapy [Paclitaxel (15 mg/kg, i.p.) followed after an interval of 30 minutes by CARBOplatin (50 mg/kg)] at five day intervals. In response to chemotherapy, tumor volume decreased approximately four-fold more in PGRMC1-deplete tumors when compared with PGRMC1-intact control tumors, suggesting that PGRMC1 promotes tumor cell viability during chemotherapeutic stress. In sum, these in vitro and in vivo findings demonstrate that PGRMC1 plays a prominent role in the growth and chemoresistance of human endometrial tumors.