BCAS2 Enhances Carcinogenic Effects of Estrogen Receptor Alpha in Breast Cancer Cells.

Estrogen receptor alpha (ERα) has an established role in breast cancer biology. Transcriptional activation by ERα is a multistep process modulated by coactivator and corepressor proteins. Breast Cancer Amplified Sequence 2 (BCAS2), is a poorly studied ERα coactivator. In this work, we characterize some of the mechanisms through which this protein increases ERα activity and how this promotes carcinogenic processes in breast cancer cells. Using protein-protein interaction and luciferase assays we show that BCAS2 interacts with ERα both in vitro and in vivo and upregulates transcriptional activation of ERα directly through its N-terminal region (AF-1) and indirectly through its C-terminal (AF-2) region, acting in concert with AF-2 interacting coactivators. Elevated expression of BCAS2 positively affects proliferation, clonogenicity and migration of breast cancer cells and directly activates ERα regulated genes which have been shown to play a role in tumor growth and progression. Finally, we used signal transduction pathway inhibitors to elucidate how BCAS2 is regulated in these cells and observed that BCAS2 is preferentially regulated by the PI3K/AKT signaling pathway. BCAS2 is an AF-1 coactivator of ERα whose overexpression promotes carcinogenic processes, suggesting an important role in the development of estrogen-receptor positive breast cancer.

Sphingolipid synthesis and role in uterine epithelia proliferation.

Sphingolipids are involved in the regulation of cell proliferation. It has been reported that diacylglycerol and sphingosine-1-phosphate generation, during the synthesis of phospho-sphingolipids, is necessary for both, G1-S transition of cell cycle during the sustained activation of protein kinase C in various cell models (MDCK, Saccharomyces and Entamoeba) and AKT pathway activation. During the estrous cycle of the rat, AKT signaling is the main pathway involved in the regulation of uterine cell proliferation. The aim of the present study was to investigate the role of sphingolipid synthesis during proliferation of uterine cells in the estrous cycle of the rat. On metestrus day, when both luminal and glandular uterine epithelia present the maximal BrdU-labeled cells (S phase cells), there was an increase in the relative abundance of total sphingomyelins, as compared to estrus day. Myriocin, a sphingolipid synthesis inhibitor administered on estrus day, before the new cell cycle of epithelial cells is initiated, decreased the abundance of sphingomyelin, accompanied by proliferation arrest in uterine epithelial cells on metestrus day. In order to study the sphingolipid signaling pathway affected by myriocin, we evaluated the activation of the PKC-AKT-GSK3b-Cyclin D3 pathway. We observed that total and phosphorylated protein kinase C diminished in uterine epithelial cells of myriocin treated animals. Interestingly, cyclin D3 nuclear localization was blocked by myriocin, concomitantly with a decrease in nuclear pRb expression. In conclusion, we demonstrate that sphingolipid synthesis and signaling are involved in uterine epithelial cell proliferation during the estrous cycle of the rat.

Oncogenic role of PinX1 in prostate cancer cells through androgen receptor dependent and independent mechanisms.

Coregulators play an important role in prostate cancer (PCa), modulating androgen receptor (AR) action and representing a possible cause of androgen deprivation therapy failure. Pin2-interacting protein X1 (PinX1) is a nucleolar protein described as a steroid hormone receptor coregulator in breast cancer cell lines. In this work, we studied the effect of PinX1 on AR action in PCa. Our results demonstrate that PinX1 acts as an AR coactivator, increasing its transcriptional activity and target gene expression, as well as proliferation, migration and colony formation in PCa cell lines. These effects are observed in the presence and absence of AR agonist and antagonists, suggesting a possible androgen independent pathway for PinX1. We present the first oncogenic roles described for PinX1, acting as a coactivator of the AR.

Breast Cancer Stem Cells and Sex Steroid Hormones.

Breast cancer stem cells (BCSCs) are a small population of tumor-initiating cells that express stem cell-associated markers. In recent years, their properties and mechanisms of regulation have become the focus of intense research due to their intrinsic resistance to conventional cancer therapies. This review describes breast cancer stem cell origin, signaling pathways involved in self-renewal, such as Wnt, Notch and Hedgehog, biomarkers linked to stemness, and the role of sex steroid hormones in BCSC regulation.

The interplay between intracellular progesterone receptor and PKC plays a key role in migration and invasion of human glioblastoma cells.

Intracellular progesterone receptors (PRs) and protein kinases C (PKCs) are known regulators of cancer cell proliferation and metastasis. Both PRs and PKCs are found overexpressed in grade IV human astrocytomas, also known as glioblastomas, which are the most frequent and aggressive brain tumors. In the present study, we investigated whether PR activation by PKC induces the migration and invasion of glioblastoma derived cell lines and if PKCα and δ isoforms are involved in PR activation. We observed that PKC activation with tetradecanoylphorbol acetate (TPA) increases the migration and invasion capacity of two human glioblastoma derived human cell lines (U251 MG and U87) and that the treatment with the PR receptor antagonist RU486 blocks these processes. Interestingly, the pharmacological inhibition of the isoenzymes PKCα and PKCδ also resulted in a blocked PR transcriptional activity. Also, TPA-dependent PR activation increases the expression of progesterone-induced blocking factor (PIBF), a known PR target gene. These results hint to an existing cross-talk between PKCs and PRs in regulating the infiltration process of human glioblastomas.

Novel role for PINX1 as a coregulator of nuclear hormone receptors.

Estrogen receptor alpha (ERα) has an established role in breast cancer biology. Transcriptional activation by ERα is a multistep process influenced by coactivator and corepressor proteins. This work shows that Pin2 interacting protein 1 (PINX1) interacts with the N-terminal domain of ERα and functions as a corepressor of ERα. Furthermore, it represses both AF-1 and AF-2 transcriptional activities. Chromatin immunoprecipitation assays verified that the interaction between ERα and PINX1 occurs on E2 regulated promoters and enhanced expression of PINX1 deregulates the expression of a number of genes that have a role in cell growth and proliferation in breast cancer. PINX1 overexpression decreases estrogen mediated proliferation of breast cancer cell lines, while its depletion shows the opposite effect. Taken together, these data show a novel molecular mechanism for PINX1 as an attenuator of estrogen receptor activity in breast cancer cell lines, furthering its role as a tumor suppressor gene in breast cancer.

PKCα and PKCδ activation regulates transcriptional activity and degradation of progesterone receptor in human astrocytoma cells.

Progesterone regulates cancer cell proliferation and invasion through its receptors (PR-A and PR-B), whose phosphorylation modifies their transcriptional activity and induce their degradation. We identified by in silico analysis a putative residue (Ser400) in PR that might be phosphorylated by protein kinase C (PKC), a family of enzymes involved in the proliferation and infiltration of astrocytomas, the most frequent and aggressive brain tumors. A grade III human astrocytoma-derived cell line was used to study the role of PKC in PR phosphorylation, transcriptional activity, and degradation. Treatment with PKC activator [tetradecanoyl phorbol acetate (TPA)] increased PR phosphorylation in Ser400 after 5 minutes, which in turn induced PR transcriptional activity and its subsequent degradation by the 26S proteasome 3-5 hours after treatment. Silencing or inhibition of PKCα and PKCδ blocked PR phosphorylation and degradation induced by TPA. Both PR isoforms were associated with PKCα and reached the maximum association after 5 minutes of TPA addition. These data correlated with immunnofluorescence assays in which nuclear colocalization of PKCα with PR increased after TPA treatment. We observed a 2-fold increase in cell proliferation after PKC activation with TPA that was reduced with the PR antagonist, RU486. The PR S400A mutant revealed that this residue is essential for PKC-mediated PR phosphorylation and degradation. Our results show a key participation of PKCα and PKCδ in PR regulation and function.

Agonistic activity of ICI 182 780 on activation of GSK 3ß/AKT pathway in the rat uterus during the estrous cycle.

We examined the ability of ICI 182,780 (ICI) to block uterine cell proliferation via protein kinase b/AKT pathway in the uterus of the rat during the estrous cycle. Intact rats, with regular estrous cycles, received a subcutaneous (s.c.) injection of either vehicle or ICI at 08:00 h on the day of proestrus or at 00:00 h on the day of estrus and sacrificed at 13:00 h of metaestrus. Estradiol (E2) and progesterone (P4) plasma levels were measured by radioimmunoassay. Both ICI treatments, induced a significant decrease (p<0.01) in uterine estrogen receptor alpha (ERα) content, had no effect on uterine progesterone receptor (PR) protein expression and caused marked nuclear localization of cyclin D1, in both luminal and glandular uterine epithelium, as compared to vehicle-treated animals. Furthermore, we detected that ICI treatment induced glycogen synthase kinase (Gsk3-ß) Ser 9 phosphorylation, which correlates with cyclin D1 nuclear localization. However, some differences were observed between the two different time schedules of administration. We observed that the administration of ICI at 08:00 h on proestrus day produced a 15% inhibition of luminal epithelial cell proliferation, reduced uterine wet weight by 21% and caused reduction of Akt phosphorylation at Ser 473 as compared to vehicle-treated animals, whereas ICI treatment at 00:00 h on estrus day had no effect on these parameters. The overall results indicate that ICI may exert agonistic and antagonistic effects on uterine cell proliferation through differential activation of the Akt pathway depending on the administration period during the estrous cycle, and indicates that the mechanism of cell proliferation during the physiological conditions of the estrous cycle, is under a different and more complex regulation than in the ovariectomized + E2 animal model.

Serotonin in testes of bat Myotis velifer during annual reproductive cycle: expression, localization, and content variations.

The mechanism of reproduction in mammals is very complex and in some cases is quite particular. For example in some bat species, the male presents a reproductive mechanism characterized by an annual testicular cycle that goes from recrudescence to regression (spermatogenesis to inactivity period, respectively). After recrudescence, the spermatozoa arrive at epididymis and wait to be expelled at the time of ejaculation during the mating period, which occurs some months later. Because serotonin (5-HT) has gained reproductive importance in the last years, the aim of the present study was to analyze the expression of this indolamine and both tryptophan hydroxylase and monoamine oxidase isoform A-enzymes involved in its metabolism-in Myotis velifer testes, a seasonal reproductive bat species that shows temporal asynchrony in its sexual cycle, across the principal periods of their reproductive cycle. By using both Falck-Hillarp histochemistry and immunofluorescence techniques, we found serotonin in vesicles of Leydig cells and probably Sertoli cells too; interestingly, both intracellular localization and concentration was variable across the different stages of the reproductive cycle, being lower during spermatogenesis phase and increasing during the mating phase. These results suggest that 5-HT is present in bat testes and it could play an important role in testicular function during their reproductive cycle.

Administration of bisphenol A to dams during perinatal period modifies molecular and morphological reproductive parameters of the offspring.

Bisphenol A (BPA) is an estrogenic agonist compound that induces changes in diverse reproductive parameters in rats. The aim of the present study was to determine the effects of BPA given in drinking water containing 10mg/L (approximate dose 1.2mg/kg BW/day), administered chronically to rats during pregnancy and lactation, on reproductive tract parameters of the offspring. 79.2% of the female offspring from BPA-treated mothers presented irregular estrous cycles. As compared to the control group, a significant increase in the thickness of the uterine epithelia and stroma was observed in the BPA group. Additionally, 60% of the female offspring from BPA mothers did not undergo abundant uterine epithelial apoptosis during the estrus phase of the cycle while control animals did. In addition, a down regulation of ERα expression was observed in epithelial cells on estrus day. The results indicate that BPA, when administered chronically in water beverages to dams, modifies the reproductive cycle of the offspring during young adulthood.

Differential expression of estrogen receptor alpha gene in the ampullae and isthmus regions of the rabbit oviduct during early pregnancy.

We characterized the expression pattern of estrogen receptor alpha (ERalpha) gene in two regions of the oviduct, ampullae and isthmus, of the rabbit (Oryctolagus cuniculus) during early pregnancy (1-4 days) by RT-PCR and immunohistochemistry. In both regions of the oviduct, ERalpha mRNA was increased (P<0.01) in pregnant rabbits as compared with non-pregnant animals (NG). In the ampullae, the greatest amount of ERalpha mRNA was detected on the third day of pregnancy (1.01+/-0.02 relative amount), followed by a decrease on Day 4. In the isthmus, an increase in ERalpha mRNA was observed during the first 4 days of pregnancy, with the greatest amount on Day 3 (1.49+/-0.3 relative amount). A marked ERalpha immunostaining was detected in epithelial, stromal and smooth muscle cells of the ampullae on the second and third days of pregnancy as compared with the NG group. In contrast, a significant increase in ERalpha immunostaining was observed on the first and second days of pregnancy with a reduction on the third and fourth days in the epithelial, stromal and smooth muscle cells of the isthmus. The overall results suggest there is differential expression pattern for the ERalpha gene in the ampullae and isthmus during early pregnancy of the rabbit, and that these variations are related to specific functions of ERalpha in the reproductive tract during early pregnancy.

Molecular mechanism of cell proliferation in rodent uterus during the estrous cycle.

The rodent uterus is a widely studied target tissue for sexual steroid hormone action. The aim of the present study was to assess the molecular mechanism that participates in the initiation of cell proliferation of the rat uterine epithelial cells during the estrus (E)-metestrus (M) transition. Cell proliferation, ERalpha, c-fos, cyclin D1 and D3, cdk4, and cdk6 proteins were assessed in these animals by immunohistochemistry. Estradiol (E(2)) and progesterone (P(4)) plasma levels were assessed by RIA. The results indicate that the glandular epithelium starts to proliferate at 21:00 h on estrus day, and initiates at least 3h before the luminal epithelium does. Fos expression was markedly increased during the afternoon of estrus day, and its increase was in parallel to ERalpha expression. Interestingly, both, cyclin D1 and D3 were abundantly expressed in the luminal and glandular epithelia, and nuclear immunolabelling of cyclin D1 and D3 precedes BrdU incorporation in the cell. cdk4 and cdk6 were localized in the nuclei in both epithelia throughout the studied time course. In addition, cdk4 was more abundant throughout estrus and metestrus days than cdk6. The overall results indicate that ERalpha, Fos and cyclins D1 and D3, cdk4 and cdk6 are expressed in both glandular and luminal epithelia of the rat uterus during the E-M transition. In conclusion, there is a good correlation between sequential expression of these proteins and cell cycle progression in the rat uterine epithelial cells during the estrous cycle. However, the differences observed in the cellular localization, time course of expression and the cellular types that express both cyclins between physiological and pharmacological conditions, demonstrated different mechanisms of regulation and should be due to the complex hormonal milieu during the estrous cycle.