Michael R. Epis

miR-331-3p Regulates ERBB-2 Expression and Androgen Receptor Signaling in Prostate Cancer

MicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression and are aberrantly expressed in human cancer. The ERBB-2 tyrosine kinase receptor is frequently overexpressed in prostate cancer and is associated with disease progression and poor survival. We have identified two specific miR-331-3p target sites within the ERBB-2 mRNA 3′-untranslated region and show that miR-331-3p expression is decreased in prostate cancer tissue relative to normal adjacent prostate tissue. Transfection of multiple prostate cancer cell lines with miR-331-3p reduced ERBB-2 mRNA and protein expression and blocked downstream phosphatidylinositol 3-kinase/AKT signaling. Furthermore, miR-331-3p transfection blocked the androgen receptor signaling pathway in prostate cancer cells, reducing activity of an androgen-stimulated prostate-specific antigen promoter and blocking prostate-specific antigen expression. Our findings provide insight into the regulation of ERBB-2 expression in cancer and suggest that miR-331-3p has the capacity to regulate signaling pathways critical to the development and progression of prostate cancer cells.

Axl Mediates Acquired Resistance of Head and Neck Cancer Cells to the Epidermal Growth Factor Receptor Inhibitor Erlotinib

Elevated expression and activity of the epidermal growth factor receptor (EGFR) is associated with development and progression of head and neck cancer (HNC) and a poor prognosis. Clinical trials with EGFR tyrosine kinase inhibitors (e.g., erlotinib) have been disappointing in HNC. To investigate the mechanisms mediating resistance to these agents, we developed an HNC cell line (HN5-ER) with acquired erlotinib resistance. In contrast to parental HN5 HNC cells, HN5-ER cells exhibited an epithelial–mesenchymal (EMT) phenotype with increased migratory potential, reduced E-cadherin and epithelial-associated microRNAs (miRNA), and elevated vimentin expression. Phosphorylated receptor tyrosine kinase profiling identified Axl activation in HN5-ER cells. Growth and migration of HN5-ER cells were blocked with a specific Axl inhibitor, R428, and R428 resensitized HN5-ER cells to erlotinib. Microarray analysis of HN5-ER cells confirmed the EMT phenotype associated with acquired erlotinib resistance, and identified activation of gene expression associated with cell migration and inflammation pathways. Moreover, increased expression and secretion of interleukin (IL)-6 and IL-8 in HN5-ER cells suggested a role for inflammatory cytokine signaling in EMT and erlotinib resistance. Expression of the tumor suppressor miR-34a was reduced in HN5-ER cells and increasing its expression abrogated Axl expression and reversed erlotinib resistance. Finally, analysis of 302 HNC patients revealed that high tumor Axl mRNA expression was associated with poorer survival (HR = 1.66, P = 0.007). In summary, our results identify Axl as a key mediator of acquired erlotinib resistance in HNC and suggest that therapeutic inhibition of Axl by small molecule drugs or specific miRNAs might overcome anti-EGFR therapy resistance. Mol Cancer Ther; 12(11); 2541–58. ©2013 AACR.

Regulation of Epidermal Growth Factor Receptor Signaling and Erlotinib Sensitivity in Head and Neck Cancer Cells by miR-7

Elevated expression and activity of the epidermal growth factor receptor (EGFR)/protein kinase B (Akt) signaling pathway is associated with development, progression and treatment resistance of head and neck cancer (HNC). Several studies have demonstrated that microRNA-7 (miR-7) regulates EGFR expression and Akt activity in a range of cancer cell types via its specific interaction with the EGFR mRNA 3′-untranslated region (3′-UTR). In the present study, we found that miR-7 regulated EGFR expression and Akt activity in HNC cell lines, and that this was associated with reduced growth in vitro and in vivo of cells (HN5) that were sensitive to the EGFR tyrosine kinase inhibitor (TKI) erlotinib (Tarceva). miR-7 acted synergistically with erlotinib to inhibit growth of erlotinib-resistant FaDu cells, an effect associated with increased inhibition of Akt activity. Microarray analysis of HN5 and FaDu cell lines transfected with miR-7 identified a common set of downregulated miR-7 target genes, providing insight into the tumor suppressor function of miR-7. Furthermore, we identified several target miR-7 mRNAs with a putative role in the sensitization of FaDu cells to erlotinib. Together, these data support the coordinate regulation of Akt signaling by miR-7 in HNC cells and suggest the therapeutic potential of miR-7 alone or in combination with EGFR TKIs in this disease.

RNA-induced silencing complex (RISC) Proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators

The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including protein kinase RNA activator (PACT), transactivation response RNA binding protein (TRBP), and Dicer, that process pre-microRNAs into mature microRNAs (miRNAs) that target specific mRNA species for regulation. There is increasing evidence for important functional interactions between the miRNA and nuclear receptor (NR) signaling networks, with recent data showing that estrogen, acting through the estrogen receptor, can modulate initial aspects of nuclear miRNA processing. Here, we show that the cytoplasmic RISC proteins PACT, TRBP, and Dicer are steroid receptor RNA activator (SRA) binding NR coregulators that target steroid-responsive promoters and regulate NR activity and downstream gene expression. Furthermore, each of the RISC proteins, together with Argonaute 2, associates with SRA and specific pre-microRNAs in both the nucleus and cytoplasm, providing evidence for links between NR-mediated transcription and some of the factors involved in miRNA processing.

The RNA-binding protein HuR opposes the repression of ERBB-2 gene expression by microRNA miR-331-3p in prostate cancer cells.

Background: The combined effect of HuR and miR-331-3p on ERBB-2 expression in prostate cancer (PCa) is unknown. Results: HuR regulated ERBB-2 expression and antagonized the repressive action of miR-331-3p. Conclusion: HuR and miR-331-3p participate in overexpression of ERBB-2 in PCa. Significance: Interplay between HuR and miR-331-3p regulates the post-transcriptional expression of ERBB-2 in PCa. ERBB-2 overexpression is associated with the development and progression of cancer and mediates its resistance to therapy. It has been suggested that post-transcriptional mechanisms control the overexpression of ERBB-2 in prostate cancer (PCa). We recently demonstrated that the 3′-untranslated region (3′-UTR) of ERBB-2 mRNA contains two specific target sites for binding of the microRNA miR-331-3p and that miR-331-3p represses ERBB-2 expression and signaling in PCa cells. Here we investigate a U-rich element situated in close proximity to the distal miR-331-3p target site in the ERBB-2 3′-UTR. Specific binding of HuR to this U-rich element promotes ERBB-2 expression in PCa cells. We show that HuR antagonizes the repressive action of miR-331-3p on its distal ERBB-2 3′-UTR target site. These results support a model in which the interplay between RNA-binding proteins and microRNAs controls the post-transcriptional regulation of gene expression and suggest that both HuR and miR-331-3p participate in the overexpression of ERBB-2 observed in some PCas.

miRNA-7-5p inhibits melanoma cell migration and invasion

Aberrant expression of microRNAs (miRNAs), a class of small non-coding regulatory RNAs, has been implicated in the development and progression of melanoma. However, the precise mechanistic role of many of these miRNAs remains unclear. We have investigated the functional role of miR-7-5p in melanoma, and demonstrate that miR-7-5p expression is reduced in metastatic melanoma-derived cell lines compared with primary melanoma cells, and that when ectopically expressed miR-7-5p significantly inhibits melanoma cell migration and invasion. Additionally, we report that insulin receptor substrate-2 (IRS-2) is a target of miR-7-5p in melanoma cells, and using RNA interference (RNAi) we provide evidence that IRS-2 activates protein kinase B (Akt), and promotes melanoma cell migration. Thus, miR-7-5p may represent a novel tumor suppressor miRNA in melanoma, acting at least in part via its inhibition of IRS-2 expression and oncogenic Akt signaling.

Erythroid defects in TRα -/- mice

Thyroid hormone and its cognate receptor (TR) have been implicated in the production of red blood cells. Here, we show mice deficient for TRalpha have compromised fetal and adult erythropoiesis. Erythroid progenitor numbers were significantly reduced in TRalpha(-/-) fetal livers, and transit through the final stages of maturation was impeded. In addition, immortalized TRalpha(-/-) erythroblasts displayed increased apoptosis and reduced capacity for proliferation and differentiation. Adult TRalpha(-/-) mice had lower hematocrit levels, elevated glucocorticoid levels, and an altered stress erythropoiesis response to hemolytic anemia. Most TRalpha(-/-) animals contained markedly altered progenitor numbers in their spleens. Strikingly, 20% of TRalpha(-/-) mice failed to elicit a stress erythropoiesis response and recovered very poorly from hemolytic anemia. We conclude that an underlying erythroid defect exists in TRalpha(-/-) mice, demon-strating the importance of TRalpha to the erythroid compartment.

miR-331-3p regulates expression of neuropilin-2 in glioblastoma

Aberrant expression of microRNAs (miRNAs), a class of small non-coding regulatory RNAs, has been implicated in the development and progression of high-grade gliomas. However, the precise mechanistic role of many miRNAs in this disease remains unclear. Here, we investigate the functional role of miR-331-3p in glioblastoma multiforme (GBM). We found that miR-331-3p expression in GBM cell lines is significantly lower than in normal brain, and that transient overexpression of miR-331-3p inhibits GBM cell line proliferation and clonogenic growth, suggesting a possible tumor suppressor role for miR-331-3p in this system. Bioinformatics analysis identified neuropilin-2 (NRP-2) as a putative target of miR-331-3p. Using transfection studies, we validated NRP-2 mRNA as a target of miR-331-3p in GBM cell lines, and show that NRP-2 expression is regulated by miR-331-3p. RNA interference (RNAi) to inhibit NRP-2 expression in vitro decreased the growth and clonogenic growth of GBM cell lines, providing further support for an oncogenic role for NRP-2 in high-grade gliomas. We also show that miR-331-3p inhibits GBM cell migration, an effect due in part to reduced NRP-2 expression. Finally, we identified a significant inverse correlation between miR-331-3p and NRP-2 expression in The Cancer Genome Atlas GBM cohort of 491 patients. Together, our results suggest that a loss of miR-331-3p expression contributes to GBM development and progression, at least in part via upregulating NRP-2 expression and increasing cell proliferation and clonogenic growth.

The Acute Box cis-Element in Human Heavy Ferritin mRNA 5′-Untranslated Region Is a Unique Translation Enhancer That Binds Poly(C)-binding Proteins

Intracellular levels of the light (L) and heavy (H) ferritin subunits are regulated by iron at the level of message translation via a modulated interaction between the iron regulatory proteins (IRP1 and IRP2) and a 5′-untranslated region. Iron-responsive element (IRE). Here we show that iron and interleukin-1β (IL-1β) act synergistically to increase H- and L-ferritin expression in hepatoma cells. A GC-rich cis-element, the acute box (AB), located downstream of the IRE in the H-ferritin mRNA 5′-untranslated region, conferred a substantial increase in basal and IL-1β-stimulated translation over a similar time course to the induction of endogenous ferritin. A scrambled version of the AB was unresponsive to IL-1. Targeted mutation of the AB altered translation; reverse orientation and a deletion of the AB abolished the wild-type stem-loop structure and abrogated translational enhancement, whereas a conservative structural mutant had little effect. Labeled AB transcripts formed specific complexes with hepatoma cell extracts that contained the poly(C)-binding proteins, iso-αCP1 and -αCP2, which have well defined roles as translation regulators. Iron influx increased the association of αCP1 with ferritin mRNA and decreased the αCP2-ferritin mRNA interaction, whereas IL-1β reduced the association of αCP1 and αCP2 with H-ferritin mRNA. In summary, the H-ferritin mRNA AB is a key cis-acting translation enhancer that augments H-subunit expression in Hep3B and HepG2 hepatoma cells, in concert with the IRE. The regulated association of H-ferritin mRNA with the poly(C)-binding proteins suggests a novel role for these proteins in ferritin translation and iron homeostasis in human liver.

Regulation of expression of deoxyhypusine hydroxylase (DOHH), the enzyme that catalyzes the activation of eIF5A, by miR-331-3p and miR-642-5p in prostate cancer cells

Background: Mechanisms underlying the regulation of DOHH expression are unclear. Results: DOHH expression is inversely associated with miR-331-3p and miR-642-5p expression in PCa cells. Transfection with miR-331-3p and miR-642-5p inhibited DOHH expression, cell proliferation and enhanced the cytotoxic effect of mimosine. Conclusion: miR-331-3p and miR-642-5p regulate DOHH expression in prostate. Significance: Restoring miR-331-3p and/or miR-642-5p to PCa may be beneficial. The enzyme deoxyhypusine hydroxylase (DOHH) catalyzes the activation of eukaryotic translation initiation factor (eIF5A), a protein essential for cell growth. Using bioinformatic predictions and reporter gene assays, we have identified a 182-nt element within the DOHH 3′-untranslated region (3′-UTR) that contains a number of target sites for miR-331-3p and miR-642-5p. Quantitative RT-PCR studies demonstrated overexpression of DOHH mRNA and underexpression of miR-331-3p and miR-642-5p in several prostate cancer cell lines compared with normal prostate epithelial cells. Transient overexpression of miR-331-3p and/or miR-642-5p in DU145 prostate cancer cells reduced DOHH mRNA and protein expression and inhibited cell proliferation. We observed synergistic growth inhibition with the combination of miR-331-3p and miR-642-5p and mimosine, a pharmacological DOHH inhibitor. Finally, we identified a significant inverse relationship between the expression of miR-331-3p or miR-642-5p and DOHH in a cohort of human prostate cancer tissues. Our results suggest a novel role for miR-331-3p and miR-642-5p in the control of prostate cancer cell growth via the regulation of DOHH expression and eIF5A activity.