Antiandrogen Flutamide-Induced Restoration of miR-449 Expression Mitigates Functional Biomarkers Associated with Ovarian Cancer Risk.

Background: The involvement of the androgen and androgen receptor (AR) pathway in the development of epithelial ovarian cancer is increasingly recognized. However, the specific mechanisms by which anti-androgen agents, such as flutamide, may prevent ovarian cancer and their efficacy remain unknown. We examined the effects of flutamide on the miRNA expression profile found in women at high risk (HR) for ovarian cancer. Methods: Ovarian and tubal tissues, free from ovarian, tubal, peritoneal cancers, and serous tubal intraepithelial carcinoma (STIC), were collected from untreated and flutamide-treated HR women. Low-risk (LR) women served as controls. Transcriptomic miRNA sequencing was performed on these 3 sample cohorts. The miRNAs that showed the most notable differential expression were subjected to functional assays in primary ovarian epithelial cells and ovarian cancer cells. Results: Flutamide treatment demonstrated a normalization effect on diminished miRNA levels in HR tissues compared to LR tissues. Particularly, the miR-449 family was significantly upregulated in HR ovarian tissues following flutamide treatment, reaching levels comparable to those in LR tissues. MiR-449a and miR-449b-5p, members of the miR-449 family, were computationally predicted to target the mRNAs of AR and colony-stimulating factor 1 receptor (CSF1R, also known as c-fms), both of which are known contributors to ovarian cancer progression, with emerging evidence also supporting their roles in ovarian cancer initiation. These findings were experimentally validated in primary ovarian epithelial cells and ovarian cancer cell lines (SKOV3 and Hey): flutamide treatment resulted in elevated levels of miR-449a and miR-449b-5p, and introducing mimics of these miRNAs reduced the mRNA and protein levels of CSF1R and AR. Furthermore, introducing miR-449a and miR-449b-5p mimics showed inhibitory effects on the migration and proliferation of ovarian cancer cells. Conclusion: Flutamide treatment restored the reduced expression of miR-449a and miR-449b-5p in HR tissues, thereby decreasing the expression of CSF1R and AR, functional biomarkers associated with an increased risk of ovarian cancer. In addition to the known direct binding of flutamide to the AR, we found that flutamide also suppresses AR expression via miR-449a and miR-449b-5p upregulation, revealing a novel dual-inhibitory mechanism on the AR pathway. Taken together, our study highlights mechanisms supporting the chemopreventive potential of flutamide in ovarian cancer, particularly in HR patients with reduced miR-449 expression.

Regulation of closely juxtaposed proto-oncogene c-fms and HMGXB3 gene expression by mRNA 3' end polymorphism in breast cancer cells.

Sense-antisense mRNA pairs generated by convergent transcription is a way of gene regulation. c-fms gene is closely juxtaposed to the HMGXB3 gene in the opposite orientation, in chromosome 5. The intergenic region (IR) between c-fms and HMGXB3 genes is 162 bp. We found that a small portion (∼4.18%) of HMGXB3 mRNA is transcribed further downstream, including the end of the c-fms gene generating antisense mRNA against c-fms mRNA. Similarly, a small portion (∼1.1%) of c-fms mRNA is transcribed further downstream, including the end of the HMGXB3 gene generating antisense mRNA against the HMGXB3 mRNA. Insertion of the strong poly(A) signal sequence in the IR results in decreased c-fms and HMGXB3 antisense mRNAs, resulting in up-regulation of both c-fms and HMGXB3 mRNA expression. miR-324-5p targets HMGXB3 mRNA 3' UTR, and as a result, regulates c-fms mRNA expression. HuR stabilizes c-fms mRNA, and as a result, down-regulates HMGXB3 mRNA expression. UALCAN analysis indicates that the expression pattern between c-fms and HMGXB3 proteins are opposite in vivo in breast cancer tissues. Together, our results indicate that the mRNA encoded by the HMGXB3 gene can influence the expression of adjacent c-fms mRNA, or vice versa.

The alternative spliced 3'-UTR mediated differential secretion of macrophage colony stimulating factor in breast cancer cells.

CSF-1 mRNA 3'UTR variants (var) are generated from alternative splicing. CSF-1 protein encoded by var-1 mRNA with long 3'UTR derived from exon-10 is rapidly secreted compared to the CSF-1 protein encoded by var-4 mRNA with short 3'UTR derived from exon-9. Secretion kinetics indicates that HuR, which binds the CSF-1 var-1 mRNA, but not var-4 mRNA, accelerates the secretion of CSF-1 protein. HuR overexpression increases the secretion rate of CSF-1 protein. In contrast, silencing of HuR does not have such an effect, suggesting other compensatory mechanisms. Effect of the CSF-1 mRNA variant 3'UTRs on cellular phenotype shows both CSF-1 var-1 or -4 mRNA is involved in the enhanced rates of migration and invasion observed by both in vitro in breast cancer cells. Our study indicates that the alternative splicing of CSF-1 mRNA 3'UTR can regulate differential secretion of CSF-1 protein.

Circulating miRNA Profiling of Women at High Risk for Ovarian Cancer.

Survival of epithelial ovarian cancer patients remains poor without significant change over many decades. There is a need to better identify women at high risk (HR) for ovarian cancer. We propose that miRNA dysregulation may play critical roles in the early stages of ovarian cancer development. Circulating miRNAs may represent an important biomarker in this context, and miRNA profiling of serum in women at HR compared to those at low risk (LR) may give insights in tumor initiation pathways. There is also rationale for a specific focus on regulation of the androgen and its related hypoxia pathways in tumor initiation. We hypothesized that subsets of these pathway related miRNAs may be downregulated in the HR state. Serum from four HR and five LR women were sequenced and analyzed for 2083 miRNAs. We found 137 miRNAs dysregulated between the HR and LR groups, of which 36 miRNAs were overexpressed in HR and the vast majority (101 miRNAs or 74%) downregulated in the HR, when compared to LR serum. mRNA targets for the differentially expressed miRNAs were analyzed from three different miRNA-mRNA interaction resources. Functional association analysis of hypoxia and androgen pathway mRNA targets of dysregulated miRNAs in HR serum revealed that all but one of the miRNAs that target 52 hypoxia genes were downregulated in HR compared to LR serum. Androgen pathway analysis also had a similar expression pattern where all but one of the miRNAs that target these 135 identified genes were downregulated in HR serum. Overall, there were 91 differentially expressed miRNA-mRNA pairings in the hypoxia analysis. In the androgen-related analysis, overall, there were 429 differentially expressed miRNA-mRNA pairs. Our pilot study suggests that almost all miRNAs that are conserved and/or validated are downregulated in the HR compared to LR serum. This study, which requires validation, suggests that, via miRNA dysregulation, involvement of both hypoxia and its related androgen pathways may contribute to the HR state. This pilot study is the first report to our knowledge that studies circulating miRNA profiling of HR and LR women.

Phenotype of vigilin expressing breast cancer cells binding to the 69 nt 3'UTR element in CSF-1R mRNA.

Vigilin, a nucleocytoplasmic shuttling protein, post-transcriptionally suppresses proto-oncogene c-fms expression (encoding CSF-1R) in breast cancer by binding to a 69 nt cis-acting 3-UTR element in CSF-1R mRNA. CSF-1R is an important mediator of breast cancer development, metastasis, and survival. We confirm that vigilin decreases in vitro reporter luciferase activity as well as the translation rate of target mRNAs. We further explore the mechanism of suppression of CSF-1R. We show that the 69 nt binding element has profound effects on translation efficiency of CSF-1R mRNA, not seen in the presence of mutation of the element. Also, mutation of the 69 nt element in the CSF-1R mRNA 3'UTR both interferes with direct vigilin binding and obviates effect of vigilin overexpression on translational repression of CSF-1R. We show that stable vigilin binding requires the full length 69 nt CSF-1R element, including the 26 nt pyrimidine-rich core. Furthermore, titration of endogenous vigilin and other proteins which bind the 69 nt element, by exogenously introduced CSF-1R mRNA 3'UTR containing the pyrimidine-rich sequence, increases the adhesion, motility, and invasion of breast cancer cells. This phenotypic effect is not seen when the 69 nt element is deleted. Lastly, we are the first to show that human breast tissues exhibit strong vigilin expression in normal breast epithelium. Our pilot data suggest decreased vigilin protein expression, along with shift from the nucleus to the cytoplasmic location, in the transition to ductal carcinoma in situ.

Human ALKBH3-induced m1A demethylation increases the CSF-1 mRNA stability in breast and ovarian cancer cells.

In ovarian and breast cancers, the actions of the cytokine CSF-1 lead to poor prognosis. CSF-1 expression can be regulated post-transcriptionally. RNA methylation is another layer of posttranscriptional regulation. The methylation of N1 atom of adenine (m1A) results in a conformational change of RNA which regulates translational efficiency. Our study indicates that the m1A is also involved in the CSF-1 mRNA decay. The alteration of ALKBH3 expression, an m1A demethylase, regulates the CSF-1 mRNA stability. Demethylation of m1A by ALKBH3 increases the half-life of CSF-1 mRNA without affecting the translation efficiency. The m1A in CSF-1 mRNA is mapped in the 5'UTR near the translation initiation site. YTHDF2, a known m6A reader which interacts with the CCR4-NOT deadenylation complex, is not the reader of m1A-containing CSF-1 mRNA. Overexpression of ALKBH3 increases CSF-1 expression and the degree of cancer cell invasiveness without affecting cell proliferation or migration. Collectively, we showed that CSF-1 mRNA decay can be regulated at an epigenetic level, and that alteration of the N1­methylation status leads to phenotypic changes in cancer cell behavior.

Expression of the cytoplasmic nucleolin for post-transcriptional regulation of macrophage colony-stimulating factor mRNA in ovarian and breast cancer cells.

The formation of the mRNP complex is a critical component of translational regulation and mRNA decay. Both the 5' and 3'UTRs of CSF-1 mRNA are involved in post-transcriptional regulation. In CSF-1 mRNA, a small hairpin loop structure is predicted to form at the extreme 5' end (2-21nt) of the 5'UTR. Nucleolin binds the hairpin loop structure in the 5'UTR of CSF-1 mRNA and enhances translation, while removal of this hairpin loop nucleolin binding element dramatically represses translation. Thus in CSF-1 mRNA, the hairpin loop nucleolin binding element is critical for translational regulation. In addition, nucleolin interacts with the 3'UTR of CSF-1 mRNA and facilitates the miRISC formation which results in poly (A) tail shortening. The overexpression of nucleolin increases the association of CSF-1 mRNA containing short poly (A)n≤26, with polyribosomes. Nucleolin both forms an mRNP complex with the eIF4G and CSF-1 mRNA, and is co-localized with the eIF4G in the cytoplasm further supporting nucleolin's role in translational regulation. The distinct foci formation of nucleolin in the cytoplasm of ovarian and breast cancer cells implicates the translational promoting role of nucleolin in these cancers.

Autocrine inhibition of the c-fms proto-oncogene reduces breast cancer bone metastasis assessed with in vivo dual-modality imaging.

Breast cancer cells preferentially home to the bone microenvironment, which provides a unique niche with a network of multiple bidirectional communications between host and tumor, promoting survival and growth of bone metastases. In the bone microenvironment, the c-fms proto-oncogene that encodes for the CSF-1 receptor, along with CSF-1, serves as one critical cytokine/receptor pair, functioning in paracrine and autocrine fashion. Previous studies concentrated on the effect of inhibition of host (mouse) c-fms on bone metastasis, with resulting decrease in osteolysis and bone metastases as a paracrine effect. In this report, we assessed the role of c-fms inhibition within the tumor cells (autocrine effect) in the early establishment of breast cancer cells in bone and the effects of this early c-fms inhibition on subsequent bone metastases and destruction. This study exploited a multidisciplinary approach by employing two non-invasive, in vivo imaging methods to assess the progression of bone metastases and bone destruction, in addition to ex vivo analyses using RT-PCR and histopathology. Using a mouse model of bone homing human breast cancer cells, we showed that an early one-time application of anti-human c-fms antibody delayed growth of bone metastases and bone destruction for at least 31 days as quantitatively measured by bioluminescence imaging and computed tomography, compared to controls. Thus, neutralizing human c-fms in the breast cancer cell alone decreases extent of subsequent bone metastasis formation and osteolysis. Furthermore, we are the first to show that anti-c-fms antibodies can impact early establishment of breast cancer cells in bone.

Nucleolin mediates microRNA-directed CSF-1 mRNA deadenylation but increases translation of CSF-1 mRNA.

CSF-1 mRNA 3'UTR contains multiple unique motifs, including a common microRNA (miRNA) target in close proximity to a noncanonical G-quadruplex and AU-rich elements (AREs). Using a luciferase reporter system fused to CSF-1 mRNA 3'UTR, disruption of the miRNA target region, G-quadruplex, and AREs together dramatically increased reporter RNA levels, suggesting important roles for these cis-acting regulatory elements in the down-regulation of CSF-1 mRNA. We find that nucleolin, which binds both G-quadruplex and AREs, enhances deadenylation of CSF-1 mRNA, promoting CSF-1 mRNA decay, while having the capacity to increase translation of CSF-1 mRNA. Through interaction with the CSF-1 3'UTR miRNA common target, we find that miR-130a and miR-301a inhibit CSF-1 expression by enhancing mRNA decay. Silencing of nucleolin prevents the miRNA-directed mRNA decay, indicating a requirement for nucleolin in miRNA activity on CSF-1 mRNA. Downstream effects followed by miR-130a and miR-301a inhibition of directed cellular motility of ovarian cancer cells were found to be dependent on nucleolin. The paradoxical effects of nucleolin on miRNA-directed CSF-1 mRNA deadenylation and on translational activation were explored further. The nucleolin protein contains four acidic stretches, four RNA recognition motifs (RRMs), and nine RGG repeats. All three domains in nucleolin regulate CSF-1 mRNA and protein levels. RRMs increase CSF-1 mRNA, whereas the acidic and RGG domains decrease CSF-1 protein levels. This suggests that nucleolin has the capacity to differentially regulate both CSF-1 RNA and protein levels. Our finding that nucleolin interacts with Ago2 indirectly via RNA and with poly(A)-binding protein C (PABPC) directly suggests a nucleolin-Ago2-PABPC complex formation on mRNA. This complex is in keeping with our suggestion that nucleolin may work with PABPC as a double-edged sword on both mRNA deadenylation and translational activation. Our findings underscore the complexity of nucleolin's actions on CSF-1 mRNA and describe the dependence of miR-130a- and miR-301a-directed CSF-1 mRNA decay and inhibition of ovarian cancer cell motility on nucleolin.

Regulation of colony stimulating factor-1 expression and ovarian cancer cell behavior in vitro by miR-128 and miR-152.

BACKGROUND: Colony stimulating factor-1 (CSF-1) plays an important role in ovarian cancer biology and as a prognostic factor in ovarian cancer. Elevated levels of CSF-1 promote progression of ovarian cancer, by binding to CSF-1R (the tyrosine kinase receptor encoded by c-fms proto-oncogene).Post-transcriptional regulation of CSF-1 mRNA by its 3' untranslated region (3'UTR) has been studied previously. Several cis-acting elements in 3'UTR are involved in post-transcriptional regulation of CSF-1 mRNA. These include conserved protein-binding motifs as well as miRNA targets. miRNAs are 21-23nt single strand RNA which bind the complementary sequences in mRNAs, suppressing translation and enhancing mRNA degradation. RESULTS: In this report, we investigate the effect of miRNAs on post-transcriptional regulation of CSF-1 mRNA in human ovarian cancer. Bioinformatics analysis predicts at least 14 miRNAs targeting CSF-1 mRNA 3'UTR. By mutations in putative miRNA targets in CSF-1 mRNA 3'UTR, we identified a common target for both miR-128 and miR-152. We have also found that both miR-128 and miR-152 down-regulate CSF-1 mRNA and protein expression in ovarian cancer cells leading to decreased cell motility and adhesion in vitro, two major aspects of the metastatic potential of cancer cells. CONCLUSION: The major CSF-1 mRNA 3'UTR contains a common miRNA target which is involved in post-transcriptional regulation of CSF-1. Our results provide the evidence for a mechanism by which miR-128 and miR-152 down-regulate CSF-1, an important regulator of ovarian cancer.

Posttranscriptional suppression of proto-oncogene c-fms expression by vigilin in breast cancer.

cis-acting elements found in 3'-untranslated regions (UTRs) are regulatory signals determining mRNA stability and translational efficiency. By binding a novel non-AU-rich 69-nucleotide (nt) c-fms 3' UTR sequence, we previously identified HuR as a promoter of c-fms proto-oncogene mRNA. We now identify the 69-nt c-fms mRNA 3' UTR sequence as a cellular vigilin target through which vigilin inhibits the expression of c-fms mRNA and protein. Altering association of either vigilin or HuR with c-fms mRNA in vivo reciprocally affected mRNA association with the other protein. Mechanistic studies show that vigilin decreased c-fms mRNA stability. Furthermore, vigilin inhibited c-fms translation. Vigilin suppresses while HuR encourages cellular motility and invasion of breast cancer cells. In summary, we identified a competition for binding the 69-nt sequence, through which vigilin and HuR exert opposing effects on c-fms expression, suggesting a role for vigilin in suppression of breast cancer progression.

Inhibition of the c-fms proto-oncogene autocrine loop and tumor phenotype in glucocorticoid stimulated human breast carcinoma cells.

The c-fms proto-oncogene encoded CSF-1 receptor and its ligand represent a feedback loop, which in a paracrine manner, is well known to promote spread of breast cancers. The role of the autocrine feedback loop in promotion of breast tumor behavior, in particular in vitro, is less well understood. The physiologic stimulation of c-fms expression by glucocorticoids (GCs) in vitro and in vivo magnifies the tumor promoting effect seen in these cells from activated c-fms signaling by CSF-1. Targeted molecular therapy against c-fms could therefore abrogate both complementary feedback loops. Using breast cancer cells endogenously co-expressing receptor and ligand, we used complementary approaches to inhibit c-fms expression and function within this autocrine pathway in the context of GC stimulation. Silencing RNA (shRNA), antisense oligonucleotide therapy (AON), and inhibition of c-fms signaling, were all used to quantitate inhibition of GC-stimulated adhesion, motility, and invasion of human breast cancer cells in vitro. shRNA to c-fms downregulated GC-stimulated c-fms mRNA by fourfold over controls, correlating with over twofold reduction in cellular invasiveness. AON therapy was also able to inhibit GC stimulation of c-fms mRNA, and resulted in threefold less invasiveness and 1.5 to 2-fold reductions in adhesion and motility. Finally, the small-molecule c-fms inhibitor Ki20227 was able to decrease in a dose-response manner, breast cancer cell invasion by up to fourfold. Inhibition of this receptor/ligand pair may have clinical utility in inhibition of the autocrine as well as the known paracrine interactions in breast cancer, thus further supporting use of targeted therapies in this disease.

Characterization of Arabidopsis AtUGT85A and AtGUS gene families and their expression in rapidly dividing tissues.

In humans, uridine 5'-diphosphate glucuronosyltransferase (UGT) operates in opposition to glucuronidase (GUS) to control activity of diverse metabolites such as hormones by reversible conjugation with glucuronic acid. Previous data revealed that, as in mammals, these enzymes are required for plant life in that a UGT from Pisum sativum (PsUGT1) controls plant development by opposing endogenous GUS activity thereby modulating the duration of the cell cycle. Here we report that a small family of genes (AtUGT85A1, 2, 3, 4, 5, and 7) homologous to pea PsUGT1 exists in the Arabidopsis genome. The AtUGT85A-encoded proteins are predicted to be membrane-associated enzymes. Three genes (AtGUS1, AtGUS2, and AtGUS3) that are homologous to a GUS-encoding gene from Scutellaria baicalensis were identified. The AtGUS-encoded proteins are predicted to be secretory (AtGUS1) and membrane-associated (AtGUS2 and AtGUS3) enzymes. Both AtUGT85A and AtGUS genes, like PsUGT1, exhibit localized, tissue-specific expression, mainly in areas of active cell division with possible involvement in cell cycle regulation.

Flavonoids: from cell cycle regulation to biotechnology.

Flavonoids have been proposed to play diverse roles in plant growth and development, including defense, symbiosis, pollen development and male fertility, polar auxin transport, and protection against ultraviolet radiation. Recently, a new role in cell cycle regulation has emerged. Genetic alteration of glucuronide metabolism by altered expression of a Pisum sativum UDP-glucuronosyltransferase (PsUGT1) results in an altered cell cycle in pea, alfalfa, and Arabidopsis. In alfalfa, altered expression of PsUGT1 results in accumulation of a flavonoid-like compound that suppresses growth of cultured cells. The results are consistent with the hypothesis that PsUGT1 functions by controlling cellular levels of a factor controlling cell cycle (FCC).

Altered life cycle in Arabidopsis plants expressing PsUGT1, a UDP-glucuronosyltransferase-encoding gene from pea.

Alfalfa (Medicago sativa) and Arabidopsis were used as model systems to examine molecular mechanisms underlying developmental effects of a microsomal UDP-glucuronosyltransferase-encoding gene from pea (Pisum sativum; PsUGT1). Alfalfa expressing PsUGT1 antisense mRNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter exhibited delayed root emergence, reduced root growth, and increased lateral root development. The timing of root emergence in wild-type and antisense plants was correlated with the transient accumulation of auxin at the site of root emergence. Cell suspension cultures derived from the antisense alfalfa plants exhibited a delay in cell cycle from 24-h in the wild-type plants to 48-h in the antisense plants. PsUGT1::uidA was introduced into Arabidopsis to demonstrate that, as in alfalfa and pea, PsUGT1 expression occurs in regions of active cell division. This includes the root cap and root apical meristems, leaf primordia, tips of older leaves, and the transition zone between the hypocotyl and the root. Expression of PsUGT1::uidA colocalized with the expression of the auxin-responding reporter DR5::uidA. Co-expression of DR5::uidA in transgenic Arabidopsis lines expressing CaMV35S::PsUGT1 revealed that ectopic expression of CaMV35S::PsUGT1 is correlated with a change in endogenous auxin gradients in roots. Roots of ecotype Columbia expressing CaMV35S::PsUGT1 exhibited distinctive responses to exogenous naphthalene acetic acid. Completion of the life cycle occurred in 4 to 6 weeks compared with 6 to 7 weeks for wild-type Columbia. Inhibition of endogenous ethylene did not correct this early senescence phenotype.

Characterization and identification of alfalfa and red clover dietary supplements using a PCR-based method.

The use of herbal remedies is very popular in the United States, with >80 million people buying plant-derived preparations that are often highly degraded or potentially contaminated with nonefficacious plant material. A method utilizing DNA-based markers to identify highly fragmented or powdered plant material sold as botanicals in dietary supplements has been developed. By incorporating and streamlining a repair reaction that utilized fill-in and ligation reactions before the PCR steps, it was possible to amplify highly degraded or sheared DNA isolated from powdered plant material removed from over-the-counter capsules. The primers for the internal transcribed spacer (ITS) region of nuclear ribosomal DNA generate a PCR fragment compatible with the sizes of the repaired DNA. Moreover, a large data set in Genbank facilitated subsequent analysis. This method is a relatively rapid and simple system to facilitate the authentication, as well as the monitoring, of the purity of botanicals in dietary supplements, even those that are improperly dried or stored.