Tyler E. Miller

MicroRNA-221/222 Confers Tamoxifen Resistance in Breast Cancer by Targeting p27Kip1

We explored the role of microRNAs (miRNAs) in acquiring resistance to tamoxifen, a drug successfully used to treat women with estrogen receptor-positive breast cancer. miRNA microarray analysis of MCF-7 cell lines that are either sensitive (parental) or resistant (4-hydroxytamoxifen-resistant (OHTR)) to tamoxifen showed significant (>1.8-fold) up-regulation of eight miRNAs and marked down-regulation (>50%) of seven miRNAs in OHTR cells compared with parental MCF-7 cells. Increased expression of three of the most promising up-regulated (miR-221, miR-222, and miR-181) and down-regulated (miR-21, miR-342, and miR-489) miRNAs was validated by real-time reverse transcription-PCR. The expression of miR-221 and miR-222 was also significantly (2-fold) elevated in HER2/neu-positive primary human breast cancer tissues that are known to be resistant to endocrine therapy compared with HER2/neu-negative tissue samples. Ectopic expression of miR-221/222 rendered the parental MCF-7 cells resistant to tamoxifen. The protein level of the cell cycle inhibitor p27Kip1, a known target of miR-221/222, was reduced by 50% in OHTR cells and by 28–50% in miR-221/222-overexpressing MCF-7 cells. Furthermore, overexpression of p27Kip1 in the resistant OHTR cells caused enhanced cell death when exposed to tamoxifen. This is the first study demonstrating a relationship between miR-221/222 expression and HER2/neu overexpression in primary breast tumors that are generally resistant to tamoxifen therapy. This finding also provides the rationale for the application of altered expression of specific miRNAs as a predictive tamoxifen-resistant breast cancer marker.

Drug-based modulation of endogenous stem cells promotes functional remyelination in vivo

Multiple sclerosis involves an aberrant autoimmune response and progressive failure of remyelination in the central nervous system. Prevention of neural degeneration and subsequent disability requires remyelination through the generation of new oligodendrocytes, but current treatments exclusively target the immune system. Oligodendrocyte progenitor cells are stem cells in the central nervous system and the principal source of myelinating oligodendrocytes. These cells are abundant in demyelinated regions of patients with multiple sclerosis, yet fail to differentiate, thereby representing a cellular target for pharmacological intervention. To discover therapeutic compounds for enhancing myelination from endogenous oligodendrocyte progenitor cells, we screened a library of bioactive small molecules on mouse pluripotent epiblast stem-cell-derived oligodendrocyte progenitor cells. Here we show seven drugs function at nanomolar doses selectively to enhance the generation of mature oligodendrocytes from progenitor cells in vitro. Two drugs, miconazole and clobetasol, are effective in promoting precocious myelination in organotypic cerebellar slice cultures, and in vivo in early postnatal mouse pups. Systemic delivery of each of the two drugs significantly increases the number of new oligodendrocytes and enhances remyelination in a lysolecithin-induced mouse model of focal demyelination. Administering each of the two drugs at the peak of disease in an experimental autoimmune encephalomyelitis mouse model of chronic progressive multiple sclerosis results in striking reversal of disease severity. Immune response assays show that miconazole functions directly as a remyelinating drug with no effect on the immune system, whereas clobetasol is a potent immunosuppressant as well as a remyelinating agent. Mechanistic studies show that miconazole and clobetasol function in oligodendrocyte progenitor cells through mitogen-activated protein kinase and glucocorticoid receptor signalling, respectively. Furthermore, both drugs enhance the generation of human oligodendrocytes from human oligodendrocyte progenitor cells in vitro. Collectively, our results provide a rationale for testing miconazole and clobetasol, or structurally modified derivatives, to enhance remyelination in patients.

Anti-microRNA-222 (Anti-miR-222) and -181B Suppress Growth of Tamoxifen-resistant Xenografts in Mouse by Targeting TIMP3 Protein and Modulating Mitogenic Signal

Background: MicroRNAs-221/222 and 181b are up-regulated in tamoxifen-resistant breast cancer. Results: Anti-miRs-222/181b regressed tamoxifen-resistant xenografts. Down-regulation of TIMP3, a common target of these miRs, facilitated growth factor signaling by regulating metalloproteases. Conclusion: Survival of tamoxifen-resistant breast cancer is dependent on miR-mediated suppression of TIMP3. Significance: Anti-miRs-222/221 and -181b can be used to render tamoxifen-resistant tumors responsive to the drug. We have shown earlier that miR-221 and -222 are up-regulated in tamoxifen-resistant MCF-7 (OHTR) cells and Her2-positive human breast tumors when compared with Her2 negative tumors. In this study, we report markedly enhanced expression of miR-181b in OHTR cells and endocrine-resistant tumors. Further, anti-miR-222 or -181b in combination with tamoxifen suppressed growth of tamoxifen-resistant xenografts in mice. Luciferase reporter assay and expression analysis showed that TIMP3, a tissue metalloproteinase inhibitor, is a common target of miR-221/222 and -181b. In situ hybridization and immunohistochemical analysis demonstrated reciprocal relationships between TIMP3 and miR-221/222/181b expression in primary human breast carcinomas. Ectopic expression of TIMP3 inhibited growth of the OHTR cells, and its depletion in MCF-7 cells reduced sensitivity to tamoxifen in vitro and in vivo. EGF-induced MAPK and AKT phosphorylation were significantly higher in OHTR cells and miR-221/222-overexpressing MCF-7 cells than in control cells, which suggests modulation of mitogenic signaling by TIMP3 and the miRs. On the contrary, phosphoMAPK and phosphoAKT levels were diminished in TIMP3-overexpressing OHTR cells and increased in TIMP3-depleted MCF-7 cells. Low levels of estrogen or tamoxifen elicited similar differences in phosphoMAPK levels in these cells. Reduced levels of TIMP3 facilitated growth of tamoxifen-resistant cells by alleviating its inhibitory effect on ADAM10 and ADAM17, which are critical for OHTR cell growth. In conclusion, miR-221/222 and -181b facilitate growth factor signaling in tamoxifen-resistant breast cancer by down-regulating TIMP3, and corresponding anti-miRs can be used to render these tumors responsive to tamoxifen.

Brain tumor stem cells: Molecular characteristics and their impact on therapy

Glioblastoma (GBM) is the most prevalent primary brain tumor and ranks among the most lethal of human cancers with conventional therapy offering only palliation. Great strides have been made in understanding brain cancer genetics and modeling these tumors with new targeted therapies being tested, but these advances have not translated into substantially improved patient outcomes. Multiple chemotherapeutic agents, including temozolomide, the first-line treatment for glioblastoma, have been developed to kill cancer cells. However, the response to temozolomide in GBM is modest. Radiation is also moderately effective but this approach is plagued by limitations due to collateral radiation damage to healthy brain tissue and development of radioresistance. Therapeutic resistance is attributed at least in part to a cell population within the tumor that possesses stem-like characteristics and tumor propagating capabilities, referred to as cancer stem cells. Within GBM, the intratumoral heterogeneity is derived from a combination of regional genetic variance and a cellular hierarchy often regulated by distinct cancer stem cell niches, most notably perivascular and hypoxic regions. With the recent emergence as a key player in tumor biology, cancer stem cells have symbiotic relationships with the tumor microenvironment, oncogenic signaling pathways, and epigenetic modifications. The origins of cancer stem cells and their contributions to brain tumor growth and therapeutic resistance are under active investigation with novel anti-cancer stem cell therapies offering potential new hope for this lethal disease.

Myosin II isoform switching mediates invasiveness after TGF-β–induced epithelial–mesenchymal transition

Despite functional significance of nonmuscle myosin II in cell migration and invasion, its role in epithelial–mesenchymal transition (EMT) or TGF-β signaling is unknown. Analysis of normal mammary gland expression revealed that myosin IIC is expressed in luminal cells, whereas myosin IIB expression is up-regulated in myoepithelial cells that have more mesenchymal characteristics. Furthermore, TGF-β induction of EMT in nontransformed murine mammary gland epithelial cells results in an isoform switch from myosin IIC to myosin IIB and increased phosphorylation of myosin heavy chain (MHC) IIA on target sites known to regulate filament dynamics (S1916, S1943). These expression and phosphorylation changes are downstream of heterogeneous nuclear ribonucleoprotein-E1 (E1), an effector of TGF-β signaling. E1 knockdown drives cells into a migratory, invasive mesenchymal state and concomitantly up-regulates MHC IIB expression and MHC IIA phosphorylation. Abrogation of myosin IIB expression in the E1 knockdown cells has no effect on 2D migration but significantly reduced transmigration and macrophage-stimulated collagen invasion. These studies indicate that transition between myosin IIC/myosin IIB expression is a critical feature of EMT that contributes to increases in invasive behavior.

Methylation of histone H3 and H4 by PRMT5 regulates ribosomal RNA gene transcription.

In an effort to understand the epigenetic regulation of ribosomal RNA gene (rDNA) expression we have previously demonstrated the role of DNA methyltransferases and methyl CpG binding proteins in rRNA synthesis. Here, we studied the role of protein arginine methyltransferase PRMT5 and the two methylated histones H3R8Me2 and H4R3Me2, in rDNA expression in Epstein Barr virus‐ transformed primary B‐cells (LCLs) and in HeLa cells responding to serum‐regulated growth. Chromatin immunoprecipitation assay showed that histones H3 and H4 associated with rRNA promoters were differentially methylated at arginine residues 8 and 3, respectively, depending on its transcriptional activity. Association of PRMT5 and methylated H3 with the unmethylated promoters in resting B‐cells was significantly reduced in rapidly growing LCLs. Unlike PRMT5 and H3R8Me2, histone H4 associated with both methylated and unmethylated rRNA promoters in resting B‐cells was methylated at the R3 residue. However, a dramatic decrease in R3 methylation of H4 recruited to the unmethylated rRNA promoters was observed in LCLs while it remained unaltered in the fraction bound to the methylated promoters. Differential interaction of PRMT5 and methylation of H3 and H4 associated with the rRNA promoters was also observed when serum starved HeLa cells were allowed to grow in serum replenished media. Ectopic expression of PRMT5 suppressed activity of both unmethylated and methylated rRNA promoter in transient transfection assay whereas siRNA mediated knockdown of PRMT5 increased rRNA synthesis in HeLa cells. These data suggest a key role of PRMT5 and the two methylated histones in regulating rRNA promoter activity. J. Cell. Biochem. 109: 553–563, 2010.

Transcription elongation factors represent in vivo cancer dependencies in glioblastoma

Glioblastoma is a universally lethal cancer with a median survival time of approximately 15 months. Despite substantial efforts to define druggable targets, there are no therapeutic options that notably extend the lifespan of patients with glioblastoma. While previous work has largely focused on in vitro cellular models, here we demonstrate a more physiologically relevant approach to target discovery in glioblastoma. We adapted pooled RNA interference (RNAi) screening technology for use in orthotopic patient-derived xenograft models, creating a high-throughput negative-selection screening platform in a functional in vivo tumour microenvironment. Using this approach, we performed parallel in vivo and in vitro screens and discovered that the chromatin and transcriptional regulators needed for cell survival in vivo are non-overlapping with those required in vitro. We identified transcription pause–release and elongation factors as one set of in vivo-specific cancer dependencies, and determined that these factors are necessary for enhancer-mediated transcriptional adaptations that enable cells to survive the tumour microenvironment. Our lead hit, JMJD6, mediates the upregulation of in vivo stress and stimulus response pathways through enhancer-mediated transcriptional pause–release, promoting cell survival specifically in vivo. Targeting JMJD6 or other identified elongation factors extends survival in orthotopic xenograft mouse models, suggesting that targeting transcription elongation machinery may be an effective therapeutic strategy for glioblastoma. More broadly, this study demonstrates the power of in vivo phenotypic screening to identify new classes of ‘cancer dependencies’ not identified by previous in vitro approaches, and could supply new opportunities for therapeutic intervention.

An epigenetic gateway to brain tumor cell identity.

Precise targeting of genetic lesions alone has been insufficient to extend brain tumor patient survival. Brain cancer cells are diverse in their genetic, metabolic and microenvironmental compositions, accounting for their phenotypic heterogeneity and disparate responses to therapy. These factors converge at the level of the epigenome, representing a unified node that can be disrupted by pharmacologic inhibition. Aberrant epigenomes define many childhood and adult brain cancers, as demonstrated by widespread changes to DNA methylation patterns, redistribution of histone marks and disruption of chromatin structure. In this Review, we describe the convergence of genetic, metabolic and microenvironmental factors on mechanisms of epigenetic deregulation in brain cancer. We discuss how aberrant epigenetic pathways identified in brain tumors affect cell identity, cell state and neoplastic transformation, as well as addressing the potential to exploit these alterations as new therapeutic strategies for the treatment of brain cancer.

Lineage-specific splicing of a brain-enriched alternative exon promotes glioblastoma progression

Tissue-specific alternative splicing is critical for the emergence of tissue identity during development, yet the role of this process in malignant transformation is undefined. Tissue-specific splicing involves evolutionarily conserved, alternative exons that represent only a minority of the total alternative exons identified. Many of these conserved exons have functional features that influence signaling pathways to profound biological effect. Here, we determined that lineage-specific splicing of a brain-enriched cassette exon in the membrane-binding tumor suppressor annexin A7 (ANXA7) diminishes endosomal targeting of the EGFR oncoprotein, consequently enhancing EGFR signaling during brain tumor progression. ANXA7 exon splicing was mediated by the ribonucleoprotein PTBP1, which is normally repressed during neuronal development. PTBP1 was highly expressed in glioblastomas due to loss of a brain-enriched microRNA (miR-124) and to PTBP1 amplification. The alternative ANXA7 splicing trait was present in precursor cells, suggesting that glioblastoma cells inherit the trait from a potential tumor-initiating ancestor and that these cells exploit this trait through accumulation of mutations that enhance EGFR signaling. Our data illustrate that lineage-specific splicing of a tissue-regulated alternative exon in a constituent of an oncogenic pathway eliminates tumor suppressor functions and promotes glioblastoma progression. This paradigm may offer a general model as to how tissue-specific regulatory mechanisms can reprogram normal developmental processes into oncogenic ones.

Targeting glioma stem cells through combined BMI1 and EZH2 inhibition

Glioblastomas are lethal cancers defined by angiogenesis and pseudopalisading necrosis. Here, we demonstrate that these histological features are associated with distinct transcriptional programs, with vascular regions showing a proneural profile, and hypoxic regions showing a mesenchymal pattern. As these regions harbor glioma stem cells (GSCs), we investigated the epigenetic regulation of these two niches. Proneural, perivascular GSCs activated EZH2, whereas mesenchymal GSCs in hypoxic regions expressed BMI1 protein, which promoted cellular survival under stress due to downregulation of the E3 ligase RNF144A. Using both genetic and pharmacologic inhibition, we found that proneural GSCs are preferentially sensitive to EZH2 disruption, whereas mesenchymal GSCs are more sensitive to BMI1 inhibition. Given that glioblastomas contain both proneural and mesenchymal GSCs, combined EZH2 and BMI1 targeting proved more effective than either agent alone both in culture and in vivo, suggesting that strategies that simultaneously target multiple epigenetic regulators within glioblastomas may be effective in overcoming therapy resistance caused by intratumoral heterogeneity.