Browsing Chester Medical School by Publisher "NCRI Cancer Conference 2014 Abstracts"
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GAS5 lncRNA Modulates the Action of mTOR Inhibitors in Prostate Cancer CellsBackground 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 – towards oligonucleotide therapies?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.
Regulation of the cell cycle and cell death by protein phosphatase 4 in breast cancer cell linesBackground At the molecular level, cell death is often regulated by the level of phosphorylation of particular proteins, i.e. by the balance of between opposing kinase and phosphatase activities on those proteins. Protein phosphatase 4 (PP4) is a PP2A-related serine/threonine phosphatase. PP2A has already been implicated in the control of cell proliferation, cell cycle and tumorigenesis. Using a functional expression cloning strategy, we have previously identified the catalytic subunit of PP4 (PP4c) as an important gene influencing the regulation of both apoptosis and cell proliferation in human leukaemic cell lines and in normal lymphocytes. The aims of this study were to examine the effects of PP4c overexpression and silencing on the cell death and survival of breast cancer cell lines. Method MCF7 and MDA-MB-231 cells were transfected with pcDNA3.1 encoding PP4c (pcDNA3-PP4c) or siRNAs to different PP4c sequences. Cells transfected with scrambled siRNA or empty vector were considered as controls. Culture viability, apoptosis and cell cycle were assessed post transfection. Results In MCF7 and metastatic MDA-MB-231 cells, PP4c over-expression exerted an inhibitory effect on cell proliferation, enhanced spontaneous apoptosis and decreased their colony forming ability. Conversely, siRNA mediated silencing of PP4 enhanced the proliferation and survival of MCF7 and MDA-MB-231 cells, affected cell cycle kinetics by enhancing the proportion of cells in S and G2/M phases, increased the colony forming ability and stimulated the anchorage independent growth. Conclusion PP4c promotes cell death and inhibits proliferation in breast cells, suggestive of a role of PP4c as tumour suppressor gene. Down regulation of PP4c expression increases cell survival, proliferation and anchorage independent growth of breast cancer cells, indicating a potential link between the PP4c expression levels, tumorigenesis and metastasis.