Nanog interaction with the androgen receptor signaling axis induce ovarian cancer stem cell regulation: studies based on the CRISPR/Cas9 system.
ABSTRACT: BACKGROUND:Ovarian cancer stem cells (OCSCs) contribute to the poor prognosis of ovarian cancer. Involvement of the androgen receptor (AR) in the malignant behaviors of other tumors has been reported. However, whether AR associates with Nanog (a stem cell marker) and participates in OCSC functions remain unclear. In this study, we investigated the interaction of Nanog with AR and examined whether this interaction induced stem-like properties in ovarian cancer cells. METHODS:AR and Nanog expression in ovarian tumors was evaluated. Using the CRISPR/Cas9 system, we constructed a Nanog green fluorescent protein (GFP) marker cell model to investigate the expression and co-localization of Nanog and AR. Then, we examined the effect of androgen on the Nanog promoter in ovarian cancer cell lines (A2780 and SKOV3). After androgen or anti-androgen treatment, cell proliferation, migration, sphere formation, colony formation and tumorigenesis were assessed in vitro and in vivo. RESULTS:Both AR and Nanog expression were obviously high in ovarian tumors. Our results showed that Nanog expression was correlated with AR expression. The androgen 5?-dihydrotestosterone (DHT) activated Nanog promoter transcription. Meanwhile, Nanog GFP-positive cells treated with DHT exhibited higher levels of proliferation, migration, sphere formation and colony formation. We also observed that the tumorigenesis of Nanog GFP-positive cells was significantly higher than that of the GFP-negative cells. Xenografts of Nanog GFP-positive cells showed significant differences when treated with androgen or anti-androgen drugs in vivo. CONCLUSIONS:The interaction of Nanog with the AR signaling axis might induce or contribute to OCSC regulation. In addition, androgen might promote stemness characteristics in ovarian cancer cells by activating the Nanog promoter. This finding merits further study because it may provide a new understanding of OCSC regulation from a hormone perspective and lead to the reevaluation of stem cell therapy for ovarian cancer.
Project description:Previous studies have shown aberrant expression of miR-214 in human malignancy. Elevated miR-214 is associated with chemoresistance and metastasis. In this study, we identified miR-214 regulation of ovarian cancer stem cell (OCSC) properties by targeting p53/Nanog axis. Enforcing expression of miR-214 increases, whereas knockdown of miR-214 decreases, OCSC population and self-renewal as well as the Nanog level preferentially in wild-type p53 cell lines. Furthermore, we found that p53 is directly repressed by miR-214 and that miR-214 regulates Nanog through p53. Expression of p53 abrogated miR-214-induced OCSC properties. These data suggest the critical role of miR-214 in OCSC via regulation of the p53-Nanog axis and miR-214 as a therapeutic target for ovarian cancer.
Project description:Hepatocellular carcinoma (HCC) is one of the most common and malignant cancers. The HCC incidence gets a strong sexual dimorphism as men are the major sufferers in this disaster. Although several studies have uncovered the presentative correlation between the axis of androgen/androgen receptor (AR) and HCC incidence, the mechanism is still largely unknown. Cancer stem cells (CSCs) are a small subgroup of cancer cells contributing to multiple tumors malignant behaviors, which play an important role in oncogenesis of various cancers including HCC. However, whether androgen/AR axis involves in regulation of HCC cells stemness remains unclear. Our previous study had identified that the pluripotency factor Nanog is not only a stemness biomarker, but also a potent regulator of CSCs in HCC. In this study, we revealed androgen/AR axis can promote HCC cells stemness by transcriptional activation of Nanog expression through directly binding to its promoter. In HCC tissues, we found that AR expression was abnormal high and got correlation with Nanog. Then, by labeling cellular endogenous Nanog with green fluorescent protein (GFP) through CRISPR/Cas9 system, it verified the co-localization of AR and Nanog in HCC cells. With in vitro experiments, we demonstrated the axis can promote HCC cells stemness, which effect is in a Nanog-dependent manner and through activating its transcription. And the xenografted tumor experiments confirmed the axis effect on tumorigenesis facilitation in vivo. Above all, we revealed a new sight of androgen/AR axis roles in HCC and provided a potential way for suppressing the axis in HCC therapy.
Project description:The Androgen Receptor (AR) is a nuclear hormone receptor that functions as a critical oncogene in all stages of prostate cancer progression, including progression to castration-resistance following androgen-deprivation therapy. Thus, identifying and targeting critical AR-regulated genes is one potential method to block castration-resistant cancer proliferation. Of particular importance are transcription factors that regulate stem cell pluripotency; many of these genes are emerging as critical oncogenes in numerous tumor cell types. Of these, Nanog has been previously shown to increase the self-renewal and stem-like properties of prostate cancer cells. Thus, we hypothesized that Nanog is a candidate AR target gene that may impart castration-resistance.We modulated AR signaling in LNCaP prostate cancer cells and assayed for Nanog expression. Direct AR binding to the NANOG promoter was tested using AR Chromatin Immunoprecipation (ChIP) and analyses of publically available AR ChIP-sequencing data-sets. Nanog over-expressing cells were analyzed for cell growth and cytotoxicity in response to the AR antagonist enzalutamide and the microtubule stabilizing agent docetaxel.AR signaling upregulates Nanog mRNA and protein. AR binds directly to the NANOG promoter, and was not identified within 75?kb of the NANOGP8 pseudogene, suggesting the NANOG gene locus was preferentially activated. Nanog overexpression in LNCaP cells increases overall growth, but does not increase resistance to enzalutamide or docetaxel.Nanog is a novel oncogenic AR target gene in prostate cancer cells, and stable expression of Nanog increases proliferation and growth of prostate cancer cells, but not resistance to enzalutamide or docetaxel.
Project description:Androgens play a major role in the regulation of normal ovarian function; however, they are also involved in the development of ovarian pathologies. These contrasting effects may involve a differential response of granulosa cells to the androgens testosterone (T) and dihydrotestosterone (DHT). To determine the molecular pathways that mediate the distinct effects of T and DHT, we studied the expression of the liver receptor homolog 1 (LRH-1) gene, which is differentially regulated by these steroids. We found that although both T and DHT stimulate androgen receptor (AR) binding to the LRH-1 promoter, DHT prevents T-mediated stimulation of LRH-1 expression. T stimulated the expression of aryl hydrocarbon receptor (AHR) and its interaction with the AR. T also promoted the recruitment of the AR/AHR complex to the LRH-1 promoter. These effects were not mimicked by DHT. We also observed that the activation of extracellular regulated kinases by T is required for AR and AHR interaction. In summary, T, but not DHT, stimulates AHR expression and the interaction between AHR and AR, leading to the stimulation of LRH-1 expression. These findings could explain the distinct response of granulosa cells to T and DHT and provide a molecular mechanism by which DHT negatively affects ovarian function.
Project description:We have cloned a cDNA coding for a novel steroid receptor co-activator protein termed SRAP from a rat prostate library. Although the nucleotide sequence of the SRAP has 78.2% identity to that of the human steroid receptor RNA activator (SRA), a novel RNA molecule which was reported to act as an RNA transcript without being translated into protein [Lanz, McKenna, Onate, Albrecht, Wong, Tsai, Tsai and O'Malley (1999) Cell 97, 17-27], the cDNA of SRAP is capable of generating a functional protein. Glutathione S-transferase pull-down assays showed that SRAP associates with the partial androgen receptor (AR) protein composed of a DNA-binding domain and an activation function 2. Luciferase assays demonstrated that SRAP enhances the transactivation activity of the AR, the glucocorticoid receptor and the peroxisome proliferator-activated receptor gamma(1) in a ligand-dependent manner. Using a green fluorescent protein (GFP) fusion-protein construct, we demonstrated in vivo translation of the GFP-SRAP fusion protein in HeLa cells co-transfected with pSG5AR and reporter gene in the presence of 5 alpha-dihydrotestosterone (DHT). Co-transfection of the GFP-SRAP fusion protein expression plasmid enhanced the transactivation activity of AR whereas incorporation of mutations in SRAP of the fusion protein resulted in loss of enhancement of the transactivation activity. Northern blot analysis and reverse transcriptase PCR assays showed that SRAP and SRA are expressed in rat and human prostate cancer cell lines respectively. In HeLa cells and the human prostate cancer cells line DU-145, co-transfected with SRAP, the DHT-dependent transactivation activities of AR were not completely inhibited by the anti-androgen flutamide, but the transactivation activities still remained high even in the presence of 5 microM flutamide, suggesting that SRAP may play an important role in enhancing AR activity in prostate cancer.
Project description:Despite androgen deprivation therapy (ADT), persistent androgen receptor (AR) signaling enables outgrowth of castration resistant prostate cancer (CRPC). In prostate cancer (PCa) cells, ADT may enhance AR activity through induction of oxidative stress. Herein, we investigated the roles of Nrf1 and Nrf2, transcription factors that regulate antioxidant gene expression, on hormone-mediated AR transactivation using a syngeneic in vitro model of androgen dependent (LNCaP) and castration resistant (C4-2B) PCa cells. Dihydrotestosterone (DHT) stimulated transactivation of the androgen response element (ARE) was significantly greater in C4-2B cells than in LNCaP cells. DHT-induced AR transactivation was coupled with higher nuclear translocation of p65-Nrf1 in C4-2B cells, as compared to LNCaP cells. Conversely, DHT stimulation suppressed total Nrf2 levels in C4-2B cells but elevated total Nrf2 levels in LNCaP cells. Interestingly, siRNA mediated silencing of Nrf1 attenuated AR transactivation while p65-Nrf1 overexpression enhanced AR transactivation. Subsequent studies showed that Nrf1 physically interacts with AR and enhances AR's DNA-binding activity, suggesting that the p65-Nrf1 isoform is a potential AR coactivator. In contrast, Nrf2 suppressed AR-mediated transactivation by stimulating the nuclear accumulation of the p120-Nrf1 which suppressed AR transactivation. Quantitative RT-PCR studies further validated the inductive effects of p65-Nrf1 isoform on the androgen regulated genes, PSA and TMPRSS2. Therefore, our findings implicate differential roles of Nrf1 and Nrf2 in regulating AR transactivation in PCa cells. Our findings also indicate that the DHT-stimulated increase in p65-Nrf1 and the simultaneous suppression of both Nrf2 and p120-Nrf1 ultimately facilitates AR transactivation in CRPC cells.
Project description:Emerging results indicate that cancer stem-like cells contribute to chemoresistance and poor clinical outcomes in many cancers, including ovarian cancer. As epigenetic regulators play a major role in the control of normal stem cell differentiation, epigenetics may offer a useful arena to develop strategies to target cancer stem-like cells. Epigenetic aberrations, especially DNA methylation, silence tumor-suppressor and differentiation-associated genes that regulate the survival of ovarian cancer stem-like cells (OCSC). In this study, we tested the hypothesis that DNA-hypomethylating agents may be able to reset OCSC toward a differentiated phenotype by evaluating the effects of the new DNA methytransferase inhibitor SGI-110 on OCSC phenotype, as defined by expression of the cancer stem-like marker aldehyde dehydrogenase (ALDH). We demonstrated that ALDH(+) ovarian cancer cells possess multiple stem cell characteristics, were highly chemoresistant, and were enriched in xenografts residual after platinum therapy. Low-dose SGI-110 reduced the stem-like properties of ALDH(+) cells, including their tumor-initiating capacity, resensitized these OCSCs to platinum, and induced reexpression of differentiation-associated genes. Maintenance treatment with SGI-110 after carboplatin inhibited OCSC growth, causing global tumor hypomethylation and decreased tumor progression. Our work offers preclinical evidence that epigenome-targeting strategies have the potential to delay tumor progression by reprogramming residual cancer stem-like cells. Furthermore, the results suggest that SGI-110 might be administered in combination with platinum to prevent the development of recurrent and chemoresistant ovarian cancer.
Project description:Post-translational degradation of protein plays an important role in cell life. We employed chimeric molecules (dihydrotestosterone-based proteolysis-targeting chimeric molecule [DHT-PROTAC]) to facilitate androgen receptor (AR) degradation via the ubiquitin-proteasome pathway (UPP) and to investigate the role of AR in cell proliferation and viability in androgen-sensitive prostate cancer cells. Western blot analysis and immunohistochemistry were applied to analyse AR levels in LNCaP cells after DHT-PROTAC treatment. Cell counting and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cell viability assay were used to evaluate cell proliferation and viability after AR elimination in both LNCaP and PC-3 cells. AR was tagged for elimination via the UPP by DHT-PROTAC, and this could be blocked by proteasome inhibitors. Degradation of AR depended on DHT-PROTAC concentration, and either DHT or an ALAPYIP-(arg)(8) peptide could compete with DHT-PROTAC. Inhibition of cell proliferation and decreased viability were observed in LNCaP cells, but not in PC-3 or 786-O cells after DHT-PROTAC treatment. These data indicate that AR elimination is facilitated via the UPP by DHT-PROTAC, and that the growth of LNCaP cells is repressed after AR degradation.
Project description:The mainstay of treatment for ovarian cancer is platinum-based cytotoxic chemotherapy. However, therapeutic resistance and recurrence is a common eventuality for nearly all ovarian cancer patients, resulting in poor median survival. Recurrence is postulated to be driven by a population of self-renewing, therapeutically resistant cancer stem cells (CSCs). A current limitation in CSC studies is the inability to interrogate their dynamic changes in real time. Here we utilized a GFP reporter driven by the NANOG-promoter to enrich and track ovarian CSCs. Using this approach, we identified a population of cells with CSC properties including enhanced expression of stem cell transcription factors, self-renewal, and tumor initiation. We also observed elevations in CSC properties in cisplatin-resistant ovarian cancer cells as compared to cisplatin-naïve ovarian cancer cells. CD49f, a marker for CSCs in other solid tumors, enriched CSCs in cisplatin-resistant and -naïve cells. NANOG-GFP enriched CSCs (GFP+ cells) were more resistant to cisplatin as compared to GFP-negative cells. Moreover, upon cisplatin treatment, the GFP signal intensity and NANOG expression increased in GFP-negative cells, indicating that cisplatin was able to induce the CSC state. Taken together, we describe a reporter-based strategy that allows for determination of the CSC state in real time and can be used to detect the induction of the CSC state upon cisplatin treatment. As cisplatin may provide an inductive stress for the stem cell state, future efforts should focus on combining cytotoxic chemotherapy with a CSC targeted therapy for greater clinical utility.
Project description:Many women with hyperandrogenemia suffer from irregular menses and infertility. However, it is unknown whether androgens directly affect reproduction. Since animal models of hyperandrogenemia-induced infertility are associated with obesity, which may impact reproductive function, we have created a lean mouse model of elevated androgen using implantation of low dose dihydrotestosterone (DHT) pellets to separate the effects of elevated androgen from obesity. The hypothalamic-pituitary-gonadal axis controls reproduction. While we have demonstrated that androgen impairs ovarian function, androgen could also disrupt neuroendocrine function at the level of brain and/or pituitary to cause infertility. To understand how elevated androgens might act on pituitary gonadotropes to influence reproductive function, female mice with disruption of the androgen receptor (Ar) gene specifically in pituitary gonadotropes (PitARKO) were produced. DHT treated control mice with intact pituitary Ar (Con-DHT) exhibit disrupted estrous cyclicity and fertility with reduced pituitary responsiveness to GnRH at the level of both calcium signaling and LH secretion. These effects were ameliorated in DHT treated PitARKO mice. Calcium signaling controls GnRH regulation of LH vesicle exotocysis. Our data implicated upregulation of GEM (a voltage-dependent calcium channel inhibitor) in the pituitary as a potential mechanism for androgen's pathological effects. These results demonstrate that gonadotrope AR, as an extra-ovarian regulator, plays an important role in reproductive pathophysiology.