• Conserved sequence-specific lincRNA-steroid receptor interactions drive transcriptional repression and direct cell fate

      Hudson, William H.; Pickard, Mark R.; de Vera, Ian M.; Kuiper, Emily G.; Mourtada-Maarabouni, Mirna; Conn, Graeme L.; Kojetin, Douglas J.; Williams, Gwyn T.; Ortlund, Eric A.; Emory University School of Medicine; Keele University; Scripps Research Institute (Nature Publishing Group, 07/11/2014)
      The majority of the eukaryotic genome is transcribed, generating a significant number of long intergenic noncoding RNAs (lincRNAs). Although lincRNAs represent the most poorly understood product of transcription, recent work has shown lincRNAs fulfill important cellular functions. In addition to low sequence conservation, poor understanding of structural mechanisms driving lincRNA biology hinders systematic prediction of their function. Here we report the molecular requirements for the recognition of steroid receptors (SRs) by the lincRNA growth arrest-specific 5 (Gas5), which regulates steroid-mediated transcriptional regulation, growth arrest and apoptosis. We identify the functional Gas5-SR interface and generate point mutations that ablate the SR-Gas5 lincRNA interaction, altering Gas5-driven apoptosis in cancer cell lines. Further, we find that the Gas5 SR-recognition sequence is conserved among haplorhines, with its evolutionary origin as a splice acceptor site. This study demonstrates that lincRNAs can recognize protein targets in a conserved, sequence-specific manner in order to affect critical cell functions.
    • GAS5 lncRNA Modulates the Action of mTOR Inhibitors in Prostate Cancer Cells

      Yacqub-Usman, Kiren; Pickard, Mark R.; Williams, Gwyn T.; Keele University, United Kingdom (NCRI Cancer Conference 2014 Abstracts, 2014)
      Background There is a need to develop new therapies for castrate-resistant prostate cancer (CRPC) and growth arrest-specific 5 (GAS5) long non-coding RNA (lncRNA), which riborepresses androgen receptor action, may offer novel opportunities in this regard. GAS5 lncRNA expression declines as prostate cancer cells acquire castrate-resistance, and decreased GAS5 expression attenuates the responses of prostate cancer cells to apoptotic stimuli. Enhancing GAS5 lncRNA expression may therefore offer a strategy to improve the effectiveness of chemotherapeutic agents. GAS5 is a member of the 5' terminal oligopyrimidine gene family, and we have therefore examined if mTOR inhibition can enhance cellular GAS5 levels in prostate cancer cells. In addition, we have determined if GAS5 lncRNA itself is required for mTOR inhibitor action in prostate cancer cells, as recently demonstrated in lymphoid cells. Method The effects of mTOR inhibitors on GAS5 lncRNA expression and cell proliferation were determined in a range of prostate cancer cell lines. Transfection of cells with GAS5 siRNA and plasmid constructs was performed to determine the involvement of GAS5 lncRNA in mTOR inhibitor action. Results Treatment with rapamycin and rapalogues increased cellular GAS5 levels and inhibited culture growth in both androgen-dependent (LNCaP) and androgen-sensitive (22Rv1) cell lines, but not in androgen-independent (PC-3 and DU145) cells. GAS5 silencing in both LNCaP and 22Rv1 cells decreased their sensitivity to growth inhibition by mTOR inhibitors. Moreover, transfection of GAS5 lncRNA sensitized PC-3 and DU145 cells to mTOR inhibitors, resulting in inhibition of culture growth. Conclusion mTOR inhibition enhances GAS5 transcript levels in some, but not all, prostate cancer cell lines. This may in part be related to endogenous levels of GAS5 expression, which tend to be lower in prostate cancer cells representative of advanced disease, particularly since current findings demonstrate a role for GAS5 lncRNA in mTOR inhibitor action in prostate cancer cells.
    • The Hormone Response Element Mimic Sequence of GAS5 LncRNA is Sufficient to Induce Apoptosis in Breast Cancer Cell Lines

      Pickard, Mark R.; Williams, Gwyn T.; Keele University, United Kingdom (2015)
      Growth arrest-specific 5 (GAS5) encodes snoRNAs and lncRNA. The latter promotes apoptosis, but its expression is down-regulated in breast cancer. The mTOR and nonsense-mediated decay pathways together regulate GAS5 transcript levels but rapalogues fail to enhance GAS5 levels in triple-negative breast cancer cells, so that mTOR inhibitor-independent induction of GAS5 may be more productive in enhancing apoptotic responses to therapies in breast cancer. Notably, GAS5 lncRNA acts by riborepression of glucocorticoid/related receptors; a stem-loop sequence constitutes the GAS5 hormone response element mimic (HREM). The aim of this study was to determine if the GAS5 HREM sequence alone is sufficient to promote the apoptosis of breast cancer cells. Cells were nucleofected with a DNA oligonucleotide corresponding to the GAS5 lncRNA HREM; controls received oligonucleotides either with scrambled GAS5 sequence or with stem complementarity present but lacking the GAS5 HRE consensus. Cells were irradiated with ultraviolet-C (UV-C) light at 20 h post-transfection to induce apoptosis. The basal apoptotic rate almost doubled in MCF7 and MDA-MB-231 cells transfected with the HREM oligonucleotide compared with controls. This effect was apparent at 20 h post¬-transfection, and a corresponding decrease was observed in culture viability; clonogenic activity was also impaired. The HREM sequence also enhanced UV-C-induced apoptosis in an additive manner in both cell lines. Endogenous GAS5 lncRNA expression was unaffected by transfection of the HREM sequence. Thus the GAS5 lncRNA HREM is sufficient to induce apoptosis in breast cancer cells, including TNBC cells and this may serve as the basis for the development of novel oligonucleotide cancer therapies. Funded by the Breast Cancer Campaign.
    • The hormone response element mimic sequence of GAS5 lncRNA is sufficient to induce apoptosis in breast cancer cell lines – towards oligonucleotide therapies?

      Pickard, Mark R.; Williams, Gwyn T.; Keele University, United Kingdom (NCRI Cancer Conference 2014 Abstracts, 2014)
      Background Growth arrest-specific 5 (GAS5), a non-protein coding gene, encodes snoRNAs and lncRNA; transcript levels are controlled by the mTOR and nonsense-mediated decay pathways. GAS5 lncRNA promotes the apoptosis of breast cells, including triple-negative breast cancer (TNBC) cells, but its expression is down-regulated in breast cancer. Rapalogues enhance GAS5 levels in oestrogen receptor-positive breast cancer cells but not in TNBC cells, so that mTOR inhibitor-independent induction of GAS5 may be more productive in enhancing apoptotic responses to therapies. Notably, GAS5 lncRNA acts by riborepression of glucocorticoid/related receptors; a stem-loop sequence constitutes the GAS5 hormone response element mimic (HREM). The aim of this study was to determine if the GAS5 HREM sequence alone is sufficient to promote the apoptosis of breast cancer cells. Method Cells were nucleofected with a DNA oligonucleotide corresponding to the GAS5 lncRNA HREM; controls received oligonucleotides either with scrambled GAS5 sequence or retaining stem-loop structure but lacking the GAS5 HRE consensus; mock-transfected cells were also studied. Cells were irradiated with ultraviolet-C (UV-C) light at 20 h post-transfection to induce apoptosis. Culture viability and apoptosis were assessed and cellular GAS5 levels were determined by RT-qPCR. Results The basal apoptotic rate almost doubled in MCF7 and MDA-MB-231 cells transfected with the HREM oligonucleotide compared with controls. This effect was apparent at 20 h post­-transfection, and a corresponding decrease was observed in culture viability. The HREM sequence also enhanced UV-C-induced apoptosis in an additive manner in both cell lines. Endogenous GAS5 lncRNA expression was unaffected by transfection of the HREM sequence. Conclusion The GAS5 lncRNA HREM is sufficient to induce apoptosis in breast cancer cells, including TNBC cells. This study serves as an exemplar of how emerging knowledge of biologically important lncRNAs may be exploited towards the development of novel oncotherapeutic agents.
    • The hormone response element mimic sequence of GAS5 lncRNA is sufficient to induce apoptosis in breast cancer cells.

      Pickard, Mark R.; Williams, Gwyn T.; Keele University (Impact Journals, 03/02/2016)
      Growth arrest-specific 5 (GAS5) lncRNA promotes apoptosis, and its expression is down-regulated in breast cancer. GAS5 lncRNA is a decoy of glucocorticoid/related receptors; a stem-loop sequence constitutes the GAS5 hormone response element mimic (HREM), which is essential for the regulation of breast cancer cell apoptosis. This preclinical study aimed to determine if the GAS5 HREM sequence alone promotes the apoptosis of breast cancer cells. Nucleofection of hormone-sensitive and -insensitive breast cancer cell lines with a GAS5 HREM DNA oligonucleotide increased both basal and ultraviolet-C-induced apoptosis, and decreased culture viability and clonogenic growth, similar to GAS5 lncRNA. The HREM oligonucleotide demonstrated similar sequence specificity to the native HREM for its functional activity and had no effect on endogenous GAS5 lncRNA levels. Certain chemically modified HREM oligonucleotides, notably DNA and RNA phosphorothioates, retained pro-apoptotic. activity. Crucially the HREM oligonucleotide could overcome apoptosis resistance secondary to deficient endogenous GAS5 lncRNA levels. Thus, the GAS5 lncRNA HREM sequence alone is sufficient to induce apoptosis in breast cancer cells, including triple-negative breast cancer cells. These findings further suggest that emerging knowledge of structure/function relationships in the field of lncRNA biology can be exploited for the development of entirely novel, oligonucleotide mimic-based, cancer therapies.
    • Molecular and Cellular Mechanisms of Action of Tumour Suppressor GAS5 LncRNA

      Pickard, Mark R.; Williams, Gwyn T.; Keele University (MDPI, 07/07/2015)
      It is increasingly recognised that lncRNAs play essential regulatory roles in fundamental biological processes and, consequently, that their dysregulation may contribute to major human diseases, including cancer. Better understanding of lncRNA biology may therefore offer new insights into pathogenetic mechanisms and thereby offer novel opportunities for diagnosis and therapy. Of particular interest in this regard is GAS5 lncRNA, which is down-regulated in multiple cancers, with expression levels related to both clinico-pathological characteristics and patient prognosis. Functional studies have further shown that GAS5 lncRNA both inhibits the proliferation and promotes the apoptosis of multiple cell types, and that together these cellular mechanisms of action are likely to form the basis of its tumour suppressor action. At the same time, advances have been made in our understanding of the molecular mechanisms of GAS5 lncRNA action in recent years, including riborepression of certain steroid hormone receptors and sequestration of miR-21, impacting key regulatory pathways of cell survival. Overall this accumulating knowledge has the potential to improve both the diagnosis and treatment of cancer, and ultimately patient outcome.
    • Regulation of apoptosis by long non-coding RNA GAS5 in breast cancer cells: implications for chemotherapy.

      Pickard, Mark R.; Williams, Gwyn T.; Keele University, United Kingdom (01/05/2014)
      The putative tumour suppressor and apoptosis-promoting gene, growth arrest-specific 5 (GAS5), encodes long ncRNA (lncRNA) and snoRNAs. Its expression is down-regulated in breast cancer, which adversely impacts patient prognosis. In this preclinical study, the consequences of decreased GAS5 expression for breast cancer cell survival following treatment with chemotherapeutic agents are addressed. In addition, functional responses of triple-negative breast cancer cells to GAS5 lncRNA are examined, and mTOR inhibition as a strategy to enhance cellular GAS5 levels is investigated. Breast cancer cell lines were transfected with either siRNA to GAS5 or with a plasmid encoding GAS5 lncRNA and the effects on breast cancer cell survival were determined. Cellular responses to mTOR inhibitors were evaluated by assaying culture growth and GAS5 transcript levels. GAS5 silencing attenuated cell responses to apoptotic stimuli, including classical chemotherapeutic agents; the extent of cell death was directly proportional to cellular GAS5 levels. Imatinib action in contrast, was independent of GAS5. GAS5 lncRNA promoted the apoptosis of triple-negative and oestrogen receptor-positive cells but only dual PI3K/mTOR inhibition was able to enhance GAS5 levels in all cell types. Reduced GAS5 expression attenuates apoptosis induction by classical chemotherapeutic agents in breast cancer cells, providing an explanation for the relationship between GAS5 expression and breast cancer patient prognosis. Clinically, this relationship may be circumvented by the use of GAS5-independent drugs such as imatinib, or by restoration of GAS5 expression. The latter may be achieved by the use of a dual PI3K/mTOR inhibitor, to improve apoptotic responses to conventional chemotherapies.
    • Targeting long non-coding RNAs (lncRNAs) with oligonucleotides in cancer therapy

      Pickard, Mark R.; Williams, Gwyn T.; University of Chester; Keele University (AME Publishing Company, 2016-10)
      No abstract