Estrogen receptor ? upregulated by lncRNA-H19 to promote cancer stem-like properties in papillary thyroid carcinoma.
ABSTRACT: Estrogen receptor ? (ER?) plays critical roles in thyroid cancer progression. However, its role in thyroid cancer stem cell maintenance remains elusive. Here, we report that ER? is overexpressed in papillary thyroid cancer stem cells (PTCSCs), whereas ablation of ER? decreases stemness-related factors expression, diminishes ALDH+ cell populations, and suppresses sphere formation ability and tumor growth. Screening estrogen-responsive lncRNAs in PTC spheroid cells, we find that lncRNA-H19 is highly expressed in PTCSCs and PTC tissue specimens, which is correlated with poor overall survival. Mechanistically, estradiol (E2) significantly promotes H19 transcription via ER? and elevates H19 expression. Silencing of H19 inhibits E2-induced sphere formation ability. Furthermore, H19 acting as a competitive endogenous RNA sequesters miRNA-3126-5p to reciprocally release ER? expression. ER? depletion reverses H19-induced stem-like properties upon E2 treatment. Appropriately, ER? is upregulated in PTC tissue specimens. Notably, aspirin attenuates E2-induced cancer stem-like traits through decreasing both H19 and ER? expression. Collectively, our findings reveal that ER?-H19 positive feedback loop has a compelling role in PTCSC maintenance under E2 treatment and provides a potential therapeutic targeting strategy for PTC.
Project description:The rearranged during transfection/papillary thyroid carcinoma (RET/PTC) tyrosine kinase is an oncogene implicated in the tumorigenesis of thyroid cancer. Recent studies by us and others have shown that RET/PTC kinase expression is induced by estrogen in breast cancer cells. Due to the critical involvement of estrogen-regulated genes in the pathogenesis of breast cancer, we investigated the expression, regulation, and function of RET/PTC kinase in breast cancer cells. We found that RET/PTC kinase expression correlates with estrogen receptor (ER) expression in breast cancer cells and tumor specimens, and that RET/PTC kinase expression is associated with a poor prognosis in ER-positive breast cancer patients. We found that estrogen rapidly induces RET/PTC kinase expression in an ER-dependent manner in breast cancer cells and that this induction is through a transcriptional regulatory mechanism. Using reporter assays, small interfering RNA (siRNA) assays, and chromatin immunoprecipitation (ChIP) assays, we demonstrated the necessity of crosstalk between ER and the forkhead box A1 (FOXA1) transcription factor in regulating RET/PTC kinase expression. In functional studies, increased expression of RET/PTC kinase induced by estrogen stimulation resulted in elevated phosphorylation of multiple downstream kinase signaling pathways. Conversely, knockdown of RET/PTC expression was associated with the inhibition of these same kinase signaling pathways, and, in fact, decreased the stimulatory effect of estrogen on the proliferation of ER-positive breast cancer cells. These results demonstrate a novel pathway of ER and FOXA1 transcription factor crosstalk in regulating RET/PTC kinase expression, and demonstrate that RET/PTC kinase is a critical regulator for the proliferation of ER-positive breast cancer cells. Taken together, our study suggests that RET/PTC kinase may serve as a novel prognostic biomarker and therapeutic target for prevention and treatment of ER-positive breast cancer.
Project description:Background:Growing evidence has indicated that the long noncoding RNA H19 (lncRNA H19), frequently deregulated in almost all tumor types tested, acted as a pivotal contributor to both cancer initiation and progression. However, the role of lncRNA H19 in human papillary thyroid carcinoma (PTC) remains controversial. The aim of the study was to investigate the expression and potential function of lncRNA H19 in human PTC. Patients and methods:The lncRNA H19 level was determined by quantitative real-time (RT)-PCR analyses in 58 PTC tissue samples and their paired paracancerous tissue samples. RNA interference, RT-PCR analysis, and Western blot assay were used to determine the impact of lncRNA H19 on epithelial-mesenchymal transition (EMT) markers in human PTC cells. The migratory and invasive capacities of PTC cells were determined by wound-healing and transwell migration and invasion assays. Results:lncRNA H19 expression was 2.417-fold higher in PTC tissues than their paired paracancerous tissue (95% CI: 1.898-2.935, P<0.0001). Higher level of lncRNA H19 was correlated to elevated expression of Vimentin, ZEB2, Twist, and Snail2. Inhibition of lncRNA H19 resulted in upregulation of E-cadherin and downregulation of Vimentin both at mRNA and protein levels. Conversely, enforced expression of the exogenous lncRNA H19 led to E-cadherin mRNA and protein downregulation and relative upregulation of Vimentin. Moreover, wound-healing and transwell migration and invasion assays showed that lncRNA H19 could promote the migratory and invasive abilities of PTC cells. Conclusion:The level of lncRNA H19 was significantly higher in PTC tissues than paired paracancerous tissue or normal tissues. Overexpression of lncRNA H19 was correlated with higher tumor burden of PTC. It also contributes to EMT process, as well as promotes migration and invasion of PTC cells.
Project description:The breast cancer stem cells (BCSC) play important roles in breast cancer occurrence, recurrence and metastasis. However, the role of estrogen signaling, a signaling pathway important in development and progression of breast cancer, in regulation of BCSC has not been well established. Previously, we identified and cloned a variant of estrogen receptor ?, ER-?36, with a molecular weight of 36 kDa. ER-?36 lacks both transactivation domains AF-1 and AF-2 of the 66 kDa full-length ER-? (ER-?66) and mediates rapid estrogen signaling to promote proliferation of breast cancer cells. In this study, we aim to investigate the function and the underlying mechanism of ER-?36-mediated rapid estrogen signaling in growth regulation of the ER-positive breast cancer stem/progenitor cells. ER-positive breast cancer cells MCF7 and T47D as well as the variants with different levels of ER-?36 expression were used. The effects of estrogen on BCSC's abilities of growth, self-renewal, differentiation and tumor-seeding were examined using tumorsphere formation, flow cytometry, indirect immunofluorence staining and in vivo xenograft assays. The underlying mechanisms were also studied with Western-blot analysis. We found that 17-?-estradiol (E2?) treatment increased the population of ER-positive breast cancer stem/progenitor cells while failed to do so in the cells with knocked-down levels of ER-?36 expression. Cells with forced expression of recombinant ER-?36, however, responded strongly to E2? treatment by increasing growth in vitro and tumor-seeding efficiency in vivo. The rapid estrogen signaling via the AKT/GSK3? pathway is involved in estrogen-stimulated growth of ER-positive breast cancer stem/progenitor cells. We concluded that ER-?36-mediated rapid estrogen signaling plays an important role in regulation and maintenance of ER-positive breast cancer stem/progenitor cells.
Project description:Although the role of estrogen signaling in breast cancer development has been extensively studied, the mechanisms that regulate the indispensable role of estrogen in normal mammary gland development have not been well studied. Because of the unavailability of culture system to maintain estrogen-receptor-positive (ER?(+)) cells in vitro, the molecular mechanisms that regulate estrogen/ER? signaling in the normal human breast are unknown. In the present study, we examined the effects of estrogen signaling on ER?(+) human luminal progenitors using a modified matrigel assay and found that estrogen signaling increased the expansion potential of these progenitors. Furthermore, we found that blocking ER? attenuated luminal progenitor expansion and decreased the luminal colony-forming potential of these progenitors. Additionally, blocking ER? decreased H19 expression in the luminal progenitors and led to the development of smaller luminal colonies. We further showed that knocking down the H19 gene in the luminal progenitors significantly decreased the colony-forming potential of the luminal progenitors, and this phenotype could not be rescued by the addition of estrogen. Lastly, we explored the clinical relevance of the estrogen-H19 signaling axis in breast tumors and found that ER?(+) tumors exhibited a higher expression of H19 as compared with ER?(-) tumors and that H19 expression showed a positive correlation with ER? expression in those tumors. Taken together, the present results indicate that the estrogen-ER?-H19 signaling axis plays a role in regulating the proliferation and differentiation potentials of the normal luminal progenitors and that this signaling network may also be important in the development of ER(+) breast cancer tumors.
Project description:Ovarian cancer is the leading cause of death in gynecological diseases. Thyroid hormone promotes proliferation of ovarian cancer cells via cell surface receptor integrin ?v?3 that activates extracellular regulated kinase (ERK1/2). However, the mechanisms are still not fully understood. Thyroxine (T4) at a physiologic total hormone concentration (10-7 M) significantly increased proliferating cell nuclear antigen (PCNA) abundance in these cell lines, as did 3, 5, 3'-triiodo-L-thyronine (T3) at a supraphysiologic concentration. Thyroid hormone (T4 and T3) treatment of human ovarian cancer cells resulted in enhanced activation of the Ras/MAPK(ERK1/2) signal transduction pathway. An MEK inhibitor (PD98059) blocked hormone-induced cell proliferation but not ER phosphorylation. Knock-down of either integrin ?v or ?3 by RNAi blocked thyroid hormone-induced phosphorylation of ERK1/2. We also found that thyroid hormone causes elevated phosphorylation and nuclear enrichment of estrogen receptor ? (ER?). Confocal microscopy indicated that both T4 and estradiol (E2) caused nuclear translocation of integrin ?v and phosphorylation of ER?. The specific ER? antagonist (ICI 182,780; fulvestrant) blocked T4-induced ERK1/2 activation, ER? phosphorylation, PCNA expression and proliferation. The nuclear co-localization of integrin ?v and phosphorylated ER? was inhibited by ICI. ICI time-course studies indicated that mechanisms involved in T4- and E2-induced nuclear co-localization of phosphorylated ER? and integrin ?v are dissimilar. Chromatin immunoprecipitation results showed that T4-induced binding of integrin ?v monomer to ER? promoter and this was reduced by ICI. In summary, thyroid hormone stimulates proliferation of ovarian cancer cells via crosstalk between integrin ?v and ER?, mimicking functions of E2.
Project description:Although long-term estrogen (E2) exposure is associated with increased breast cancer (BC) risk, and E2 appears to sustain growth of BC cells that express functional estrogen receptors (ERs), its role in promoting BC stem cells (CSCs) remains unclear. Considering that Gli1, part of the Sonic hedgehog (Shh) developmental pathway, has been shown to mediate CSCs, we investigated whether E2 and Gli1 could promote CSCs and epithelial-mesenchymal transition (EMT) in ER+ BC cell lines.We knocked down Gli1 in several BC cells using a doxycycline-controlled vector, and compared Gli1-knockdown cells and Gli1+ cells in behavior and expression of ER, Gli1, ALDH1 (BC-CSC marker), Shh, Ptch1 (Shh receptor) and SOX2, Nanog and Bmi-1 (CSC-associated transcriptions factors), using PCR; tissue microarrays, western blot; chromatin immunoprecipitation q-PCR, confocal immunofluorescence microscopy; fluorescence-activated cell sorting; annexin-flow cytometry (for apoptosis); mammosphere culture; and colony formation, immunohistochemistry, Matrigel and wound-scratch assays.Both mRNA and protein expressions of ER correlated with those of Gli1 and ALDH1. E2 induced Gli1 expression only in ER+ BC cells. E2 promoted CSC renewal, invasiveness and EMT in ER+/Gli1+ cells but not in Gli1-knockdown cells.Our results indicate that estrogen acts via Gli1 to promote CSC development and EMT in ER+ BC cells. These findings also imply that Gli1 mediates cancer stem cells, and thus could be a target of a novel treatment for ER+ breast cancer.
Project description:BACKGROUND: Through a transcriptome microarray analysis, we have isolated Anterior gradient protein 2 (AGR2) as a gene up-regulated in papillary thyroid carcinoma (PTC). AGR2 is a disulfide isomerase over-expressed in several human carcinomas and recently linked to endoplasmic reticulum (ER) stress. Here, we analyzed the expression of AGR2 in PTC and its functional role. METHODS: Expression of AGR2 was studied by immunohistochemistry and real time PCR in normal thyroids and in PTC samples. The function of AGR2 was studied by knockdown in PTC cells and by ectopic expression in non-transformed thyroid cells. The role of AGR2 in the ER stress was analyzed upon treatment of cells, expressing or not AGR2, with Bortezomib and analyzing by Western blot the expression levels of GADD153. RESULTS: PTC over-expressed AGR2 at mRNA and protein levels. Knockdown of AGR2 in PTC cells induced apoptosis and decreased migration and invasion. Ectopic expression of AGR2 in non-transformed human thyroid cells increased migration and invasion and protected cells from ER stress induced by Bortezomib. CONCLUSIONS: AGR2 is a novel marker of PTC and plays a role in thyroid cancer cell survival, migration, invasion and protection from ER stress.
Project description:Background:The incidence of papillary thyroid cancer (PTC) is increasing faster than any other solid tumors worldwide. Invasion and metastasis are the main reasons for the poor prognosis of patients with PTC. Previously, we observed significantly low expression of miRNA-299-5p in invasive PTC tissue samples. Aim:The present study aimed to determine whether miR-299-5p plays a key role in PTC migration and invasion. Materials and methods:The miR-299-5p expression level was measured using quantitative real-time PCR in 109 human PTC samples and paired adjacent normal tissues and in the human BCPAP PTC cell line. The effects of miR-299-5p on PTC cell migration and invasion were assessed using wound healing and transwell assays. In addition, we searched for the miR-299-5p target, and the potential mechanism was demonstrated using a reporter assay and rescue experiment. Results:The expression of miR-299-5p was associated with gender and extrathyroidal extension, and an elevated level of miR-299-5p suppressed BCPAP cell migration and invasion. Estrogen receptor ? (ER?) is a direct target of miR-299-5p. The expression level of ER? was significantly higher in PTC tissues and was associated with migration and invasion in PTC cells. Overexpression of ER? could impair miR-299-5p-induced inhibition of migration and invasion. As a key factor of the pathway related to PTC invasion, Gli1 can be combined with ER? and can be regulated by miR-299-5p. Conclusion:Our data suggested that miR-299-5p could participate in PTC migration and invasion and could be a potential therapeutic target for patients with aggressive PTC tumors.
Project description:BACKGROUND:Hormonal differences are hypothesized to contribute to the approximately ?2-fold higher thyroid cancer incidence rates among women compared with men worldwide. Although thyroid cancer cells express estrogen receptors and estrogen has a proliferative effect on papillary thyroid cancer (PTC) cells in vitro, epidemiologic studies have not found clear associations between thyroid cancer and female hormonal factors. We hypothesized that polymorphic variation in hormone pathway genes is associated with the risk of developing papillary thyroid cancer. METHODS:We evaluated the association between PTC and 1151 tag single nucleotide polymorphisms (SNPs) in 58 candidate gene regions involved in sex hormone synthesis and metabolism, gonadotropins, and prolactin in a case-control study of 344 PTC cases and 452 controls, frequency matched on age and sex. Odds ratios and p-values for the linear trend for the association between each SNP genotype and PTC risk were estimated using unconditional logistic regression. SNPs in the same gene region or pathway were aggregated using adaptive rank-truncated product methods to obtain gene region-specific or pathway-specific p-values. To account for multiple comparisons, we applied the false discovery rate method. RESULTS:Seven SNPs had p-values for linear trend <0.01, including four in the CYP19A1 gene, but none of the SNPs remained significant after correction for multiple comparisons. Results were similar when restricting the dataset to women. p-values for examined gene regions and for all genes combined were ?0.09. CONCLUSIONS:Based on these results, SNPs in selected hormone pathway genes do not appear to be strongly related to PTC risk. This observation is in accord with the lack of consistent associations between hormonal factors and PTC risk in epidemiologic studies.
Project description:Human thyroid cancer derived cell lines are widely used to study the mechanisms involved in thyroid carcinogenesis. However, there is limited availability of non-cross-contaminated cancer cell lines derived from papillary thyroid carcinoma (PTC), and the B-CPAP cell line is one of the few such lines. B-CPAP cells have been genetically and cytogenetically well-characterized, but details of their stemness features remain uncertain. Considering that this cell line is extensively used for in vitro studies on thyroid tumorigenesis, we broaden its functional and molecular profiles as well as the tumorigenic capacity. We used functional assays (sphere-forming capacity and efficiency), assessed self-renewal and propagation efficiency and tested in vivo tumorigenicity in Hsd:Athymic Nude-Foxn1nu mice. Expression of markers of stemness, differentiation, and epithelial-mesenchymal transition were estimated at RNA and protein levels in adherent parental cells and sphere-forming cells. Functional aspects and stemness features were compared with normal thyrocytes. Protein expression of xenograft tumors was evaluated by immunohistochemistry. B-CPAP sphere-forming cells were able to form thyrospheres theoretically indefinitely in an appropriate serum-free medium, reverting to the adherent parental cell phenotype when cultured in differentiation medium. Different expression of ALDH1-A1 and CD44 stemness markers and TTF-1 and CK19 differentiation markers allowed discrimination between isolated sphere-forming cells and adherent parental cells, indicating that sphere-forming cells retained stem-like features. In keeping with these observations, tumorigenicity assays confirmed that, relative to parental adherent cells, thyrospheres had enhanced capacity to initiate xenograft tumors. Thyrospheres from normal cell line retained very low functional capacity, as well as different stemness markers expression compared to tumor thyrospheres. Our findings may constitute a useful background to develop an in vitro model for assessing the origin and progression of papillary thyroid carcinoma bearing BRAFV600E and TERT promoter mutations.