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Regulation of apoptosis by long non-coding RNA GAS5 in breast cells: 1 2 implications for chemotherapy 3 4 5 6 7 8 9 Mark R. Pickard* & Gwyn T. Williams* 10 11 12 Apoptosis Research Group, Institute of Science and Technology in Medicine, Huxley 13 14 15 Building, School of Life Sciences, Keele University, Keele ST5 5BG, United Kingdom. 16 17 18 19 20 21 22 23 24 25 *Corresponding authors: 26 27 28 M.R.Pickard: E-mail: [email protected]; Tel: 0044 (0)1782 733671; Fax: 0044 29 30 (0)1782 733516 31 32 33 34 G.T. Williams: E-mail: [email protected]; Tel: 0044 (0)1782 733032; Fax: 0044 35 36 (0)1782 733516 37 38 39 40 41 42 43 Running title: GAS5 and breast cancer 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 1 63 64 65

1 2 Abstract 3 4 5 Purpose: The putative tumour suppressor and apoptosis-promoting , growth arrest- 6 7 8 specific 5 (GAS5), encodes lncRNA and snoRNAs. Its expression is down-regulated in breast 9 10 cancer, which adversely impacts patient prognosis. In this preclinical study, the consequences 11 12 13 of decreased GAS5 expression for breast cancer cell survival following treatment with 14 15 chemotherapeutic agents are addressed. In addition, functional responses of triple-negative 16 17 18 breast cancer cells to GAS5 lncRNA are examined, and mTOR inhibition as a strategy to 19 20 enhance cellular GAS5 levels is investigated. Methods: Breast cancer cell lines were 21 22 23 transfected with either siRNA to GAS5 or with a plasmid encoding GAS5 lncRNA and the 24 25 effects on breast cancer cell survival were determined. Cellular responses to mTOR inhibitors 26 27 were evaluated by assaying culture growth and GAS5 transcript levels. Results: GAS5 28 29 30 silencing attenuated cell responses to apoptotic stimuli, including classical chemotherapeutic 31 32 agents; the extent of cell death was directly proportional to cellular GAS5 levels. Imatinib 33 34 35 action in contrast, was independent of GAS5. GAS5 lncRNA promoted the apoptosis of 36 37 triple-negative and estrogen receptor-positive cells but only dual PI3K/mTOR inhibition was 38 39 40 able to enhance GAS5 levels in all cell types. Conclusions: Reduced GAS5 expression 41 42 attenuates apoptosis induction by classical chemotherapeutic agents in breast cancer cells, 43 44 providing an explanation for the relationship between GAS5 expression and breast cancer 45 46 47 patient prognosis. Clinically, this relationship may be circumvented by the use of GAS5- 48 49 independent drugs such as imatinib, or by restoration of GAS5 expression. The latter may be 50 51 52 achieved by the use of a dual PI3K/mTOR inhibitor, to improve apoptotic responses to 53 54 conventional chemotherapies. 55 56 57 58 59 60 61 62 2 63

64 65 Keywords: GAS5, ncRNA, apoptosis, breast, cancer, chemotherapy. 1 2 3 Abbreviations: 4 5 6 7 A490 Absorbance at 490 nm 8 9 10 CT Threshold cycle 11 12 13 EST Expressed sequence tag 14 15 16 5-FU 5-Fluorouracil 17 18 19 20 GAS5 Growth arrest-specific 5 21 22 23 HER2 Human epidermal growth factor receptor 2 24 25 26 lncRNA Long ncRNA 27 28 29 30 MCT Multiple comparison test 31 32 33 NC Negative control 34 35 36 ncRNA Non-coding RNA 37 38 39 40 NMD Nonsense-mediated decay 41 42 43 RT-qPCR Real time reverse transcription polymerase chain reaction 44 45 46 5′-TOP 5′-Terminal oligopyrimidine tract 47 48 49 50 TNBC Triple-negative breast cancer 51 52 53 UV-C Ultraviolet-C. 54 55 56 57 58 59 60 61 62 3 63

64 65 Introduction 1 2 Long ncRNAs (size range: 0.2 - 100 kb) are emerging as key regulators of fundamental 3 4 5 cellular processes and are increasingly recognized as of importance in the pathogenesis, 6 7 diagnosis and treatment of major , including breast cancer [1-5]. Apart from 8 9 ribosomal , lncRNAs comprise a plethora of mRNA-like transcripts (long intergenic 10 11 12 ncRNAs, pseudogene-derived transcripts and sequences that are intronic or antisense to 13 14 protein coding ) [1, 6, 7], which can act as signals, scaffolds, guides, decoys or tethers 15 16 17 to modulate, for example, the maintenance of subcellular structures, chromatin modification 18 19 and transcriptional/post-transcriptional regulation of gene expression [7, 8]. 20 21 22 23 24 25 26 The lncRNA and small nucleolar RNA (snoRNA) host gene, growth arrest-specific 5 27 28 (GAS5), is of particular interest in breast cancer, since its expression is down-regulated in 29 30 31 tumour tissue [9], and patient prognosis is related to GAS5 transcript levels [10]. GAS5 is a 32 33 5′-terminal oligopyrimidine tract (5′-TOP) gene, and comprises 12 exons which are 34 35 36 alternatively spliced to yield two possible mature lncRNAs [11]. GAS5 intronic sequence 37 38 additionally encodes ten distinct box C/D snoRNAs [11]; three of which (U44, U74 and U78) 39 40 may serve as miRNA precursors [12]. Multiple other GAS5 expressed sequence tags (ESTs) 41 42 43 have also been identified, many of which contain retained introns, indicating that post- 44 45 transcriptional processing of GAS5 RNA is complex. 46 47 48 49 50 51 52 GAS5 transcripts accumulate in growth-arrested cells [11, 13] due to interplay between the 53 54 mTOR pathway (through its regulation of translation of RNAs carrying the 5′-TOP sequence) 55 56 57 and the nonsense-mediated decay (NMD) pathway (due to the GAS5 short reading frame) 58 59 [11, 14]. Thus, in actively growing cells, where mTOR activity is high, translation of the 60 61 62 4 63

64 65 short reading frame is promoted, resulting in degradation of transcripts by the NMD pathway, 1 2 and consequently low cellular GAS5 lncRNA levels. Suppression of cell growth and mTOR 3 4 5 activity prevents the active translation of GAS5 transcripts and their consequent degradation 6 7 by NMD, resulting in the accumulation of GAS5 lncRNA [11, 13, 14]. Direct inhibition of 8 9 10 mTOR activity by rapamycin and rapalogues produces the predicted increase in cellular 11 12 GAS5 lncRNA [11, 15, 16]. 13 14 15 16 17 18 19 In breast and other cell types, GAS5 inhibits cell proliferation and/or stimulates apoptosis [9, 20 21 17-21], i.e., key determinants of cell survival that are often disrupted in cancer [22]. Indeed, 22 23 24 elevated GAS5 expression inhibits human breast tumour growth in a xenograft mouse model 25 26 [21], consistent with a tumour suppressor role for this gene. Moreover, the apoptosis- 27 28 promoting activity of GAS5 has implications for cancer therapies, since the action of many 29 30 31 chemotherapeutic drugs ultimately depends on engagement of the cellular apoptotic 32 33 machinery [23, 24]. However, the consequences of reduced GAS5 expression for 34 35 36 chemotherapeutic drug action in breast cancer cells have not been examined to-date. 37 38 39 40 41 42 In this study we have focussed on the chemotherapeutic implications of reduced GAS5 43 44 45 expression in breast cancer. Firstly, we have investigated the hypothesis that GAS5 silencing 46 47 impairs the responses of breast cancer cells to apoptotic stimuli. Secondly, we have examined 48 49 50 whether functional responses to GAS5 remain intact in triple-negative breast cancer (TNBC) 51 52 cells. Finally, we have investigated mTOR inhibition as a strategy to enhance cellular GAS5 53 54 55 levels in breast cancer cells and consequently to suppress cancer growth through the 56 57 induction of apoptosis and/or cell cycle arrest. 58 59 60 61 62 5 63

64 65

1 2 3 Materials and methods 4 5 6 7 Material 8 9 10 Cell lines were from ATCC-LGC Promochem (Teddington, UK). Cell culture materials and 11 12 classical chemotherapeutic drugs were from Sigma-Aldrich Company Ltd (Gillingham, UK). 13 14 15 Sources of other drugs were: rapamycin (Millipore, Watford, UK); temsirolimus and 16 17 everolimus (LC Laboratories, Woburn, MA); BEZ235 and AZD8055 (Stratech Scientific, 18 19 20 Newmarket, UK); and imatinib (mesylate) (Cayman Chemical, Ann Arbor, MI). TRIzol, 21 22 reverse transcription reagents, TaqMan assays and Silencer Select siRNAs were from Life 23 24 Technologies Ltd (Paisley, UK). SensiFast Probe Hi-ROX kit was from Bioline (London, 25 26 27 UK), RQ1 RNase-free DNase and the MTS assay (CellTiter 96 AQueous One Solution Cell 28 29 Proliferation Assay) were from Promega (Southampton, UK), RNAiFect was from Qiagen 30 31 32 (Crawley, UK) and nucleofector solution V was from Lonza Biosciences (Verviers, 33 34 Belgium). 35 36 37 38 39 40 41 Methods 42 43 44 Cell culture 45 46 47 The breast cancer cell lines MCF7, T-47D and MDA-MB-231 were generated from 48 49 50 secondary stocks of cells which had been frozen down within two weeks receipt from the 51 52 ATCC. Cells were cultured in R-10 medium (RPMI-1640 supplemented with 2 mM L- 53 54 55 glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 10% fetal bovine serum and 50 µg/ml 56 57 gentamicin) at 37 °C in a humidified incubator with 5% CO2. Cell lines were replaced with 58 59 60 fresh stocks after a maximum culture period of two months. 61 62 6 63

64 65

1 2 3 RNA interference by siRNA 4 5 6 7 Cells were transfected with Ambion Silencer Select siRNAs using RNAiFect reagent, 8 9 according to a standard protocol [20]. Up to 4 different GAS5 siRNAs were employed, 10 11 termed siRNAs #1 – 4 (product codes/targeted exons are: n272334/exon 2; n272337/exon 8; 12 13 14 n272340/exon 12 and n272331/exon 12, respectively); controls received negative control 15 16 (NC) siRNA (code AM4611). 17 18 19 20 21 22 23 Plasmid DNA transfection 24 25 26 Plasmids were: pcDNA3/GAS5.O1 (encodes GAS5 O1 EST); pcDNA3/GAS5.AE (encodes 27 28 29 mature GAS5 lncRNA); and pcDNA3 vector (for controls) [20]. Cells were nucleofected 30 31 with plasmids (2 µg per 2 x 106 cells in 0.1 ml nucleofector solution V) using programmes E- 32 33 34 014 and X-013 for MCF7 and MDA-MB-231, respectively, and cells were plated in 3 ml R- 35 36 10 medium in 6-well plates. 37 38 39 40 41 42 43 Induction of cell death and cell survival assays 44 45 46 At 72 h post-siRNA transfection or at 20 h post-plasmid nucleofection, cells were 47 48 trypsinized, sampled for RNA, then seeded (0.8 x 105 cells for siRNA-transfected cells; 1.6 x 49 50 5 51 10 cells for plasmid-transfected cells) into 12-well plates. Ultraviolet-C (UV-C) irradiation 52 53 was performed prior to plating [20]; doses were 40 J/m2 for MCF7 and MDA-MB-231 cells, 54 55 2 56 and 60 J/m for T-47D cells; controls were mock-irradiated. For drug treatments, cells were 57 58 cultured for 20 h, before addition of 5-fluorouracil (5-FU; 175 µM), docetaxel (10 µM), 59 60 61 62 7 63

64 65 imatinib (50 µM) or vehicle (0.25% dimethyl sulphoxide). Cells were cultured for 48 h post- 1 2 treatment, then adherent cells were trypsinized and combined with non-adherent cells for 3 4 5 analysis of cell survival. 6 7 8 9 10 11 Apoptosis was routinely determined by assessment of nuclear morphology by fluorescence 12 13 14 microscopy after staining with acridine orange (25 µg/ml); cells containing condensed or 15 16 fragmented chromatin were scored as apoptotic. Cell viability was determined by counting of 17 18 19 nigrosin blue (0.1% (w/v)) stained samples using a haemocytometer and light microscopy. 20 21 For clonogenic assays, cells were re-plated in culture medium supplemented with 10% (v/v) 22 23 24 cell-conditioned medium in 6-well plates, cultured for 3 weeks, then the number of colonies 25 26 was counted after staining with crystal violet. 27 28 29 30 31 32 33 Assessment of mTOR inhibitor sensitivity 34 35 36 Cells were plated in 96-well plates (500 cells in 0.1 ml R10 medium) and in 12-well plates 37 38 (1.6 x 105 cells in 0.8 ml R10 medium). After 4 h, an equal volume of medium containing the 39 40 41 appropriate drug or vehicle (final concentrations: 1 µM rapamycin, 10 nM everolimus, 10 nM 42 43 temsirolimus, 100 nM BEZ235, 50 nM AZD8055 or 0.1% dimethyl sulphoxide) was added. 44 45 46 Experiments were terminated after a further 20 h (12-well plates; for GAS5 determination) or 47 48 70 h (96-well plates; for MTS assay). For the latter, sample absorbance readings at 490 nm 49 50 51 (A490) were corrected for the appropriate medium plus drug blank values. 52 53 54 55 56 57 58 59 60 61 62 8 63

64 65 Effect of cell density-induced growth arrest on cellular GAS5 levels 1 2 3 For low cell density, logarithmic phase cultures, cells were plated at 2 x 105 per well (6-well 4 5 6 plate) then harvested after 24 h culture. For high cell density, stationary phase cultures, cells 7 8 plated at 10 x 105 cells per well (6-well plate) then harvested after 96 h culture. 9 10 11 12 13 14 15 Real time RT-PCR (RT-qPCR) 16 17 18 TaqMan Gene Expression Assays (assay codes Hs99999901_m1 for 18S and 19 20 Hs03464472_m1 for GAS5) were employed with cDNA prepared by random hexamer 21 22 23 priming, as described previously [20]. Assays (25 µl) contained 10 ng sample cDNA or 0.2 – 24 25 60 ng standard cDNA (prepared from MCF7 cells). Input amounts of samples were calculated 26 27 28 from their respective threshold cycle (CT) values, using standard curves generated with each 29 30 assay. Data were expressed relative to 18S rRNA. 31 32 33 34 35 36 37 Statistical analyses 38 39 40 Data are presented as the mean ± SEM; the number of observations (n) refers to different 41 42 43 transfected samples (each from separate cultures) or separate cultures. Data analysis was 44 45 either by an unpaired Student's t-test (when comparing two groups only) or by one-way 46 47 analysis of variance with either Bonferroni's multiple comparison test (MCT) or Dunnett’s 48 49 50 MCT (when comparing multiple groups versus a single group), as indicated. Homogeneity of 51 52 variance was checked by Bartlett's test and, where necessary, data were transformed (log or 53 54 55 square root) prior to analysis. The relationship between survival parameters and GAS5 levels 56 57 in cells was analyzed by linear regression. Statistical analyses were performed using 58 59 60 GraphPad Prism v4.03. 61 62 9 63

64 65

1 2 3 Results 4 5 6 7 GAS5 silencing attenuates apoptosis induction in breast cancer cells 8 9 10 To examine the effects of reduced GAS5 expression on breast cancer cell survival, GAS5 11 12 siRNAs were employed to silence endogenous GAS5 expression in two cell lines, MCF7 and 13 14 15 T-47D; a range of siRNAs were employed to reduce the likelihood of ‘off-target’ effects. The 16 17 influence of GAS5 silencing on cell survival was examined under basal conditions and after 18 19 20 apoptosis induction by a range of stimuli. 21 22 23 24 25 26 In MCF7 cells, GAS5 siRNAs reduced GAS5 transcript levels by upto one third of control 27 28 29 levels (Fig. 1a). This had no statistically significant effect on basal apoptosis (Fig. 1b), short- 30 31 term viability (Fig. 1d), viable cell number (Fig. 1f) or clonogenic activity (Fig. 1h), as 32 33 34 judged from mock-irradiated cells. However, GAS5 knockdown attenuated apoptosis 35 36 induction by UV-C irradiation (Fig. 1c) and the consequent reduction in culture viability (Fig. 37 38 1e) and clonogenic activity (Fig. 1i). Growth arrest due to UV-C treatment appeared less 39 40 41 markedly affected however, as judged from viable cell counts (Fig. 1g). 42 43 44 45 46 47 Consistent with these observations, GAS5 knockdown in T-47D cells (Fig. 2a) diminished 48 49 50 apoptosis induction by UV-C irradiation (Fig. 2c), as well as the associated losses in culture 51 52 viability (Fig. 2e) and clonogenic activity (Fig. 2i); less robust effects on UV-C-induced 53 54 55 growth arrest were also noted for this cell line (Fig. 2g). Basal apoptosis was also reduced by 56 57 GAS5 silencing in T-47D cells (Fig. 2b), in contrast to findings in MCF7 cells (Fig. 1b). This 58 59 60 61 62 10 63

64 65 was associated with increased culture viability (Fig. 2d), but basal clonogenic activity was 1 2 unaffected (Fig. 2h). 3 4 5 6 7 8 9 These findings were confirmed (Figs. 3a & b) in control experiments examining the effects of 10 11 GAS5 silencing on chemotherapeutic drug action. GAS5 silencing diminished docetaxel- 12 13 14 induced apoptosis and the associated loss of culture viability in both MCF7 (Fig. 3c) and T- 15 16 47D cells (Fig. 3d). Similar effects were observed regarding 5-FU action in MCF7 (Fig. 3e) 17 18 19 and T-47D (Fig. 3f) cells, whereas, imatinib action was unaffected by GAS5 silencing (Figs. 20 21 3g & h). 22 23 24 25 26 27 28 Breast cancer cell survival is related to cellular GAS5 levels 29 30 31 If impaired responses to chemotherapeutic drugs underlie the clinical relationship between 32 33 34 patient survival and breast cancer GAS5 levels [10], then parameters of breast cancer cell 35 36 survival should show quantitative relationships with cellular GAS5 levels. Regression 37 38 analysis was therefore performed on data from the GAS5 silencing experiments; cellular 39 40 41 GAS5 levels were expressed relative to those in negative control siRNA-treated cultures to 42 43 facilitate comparisons between the two cell lines. 44 45 46 47 48 49 50 Under basal conditions, apoptosis and viability were related to cellular GAS5 levels in T-47D 51 52 cells (Fig. 4b) but not in MCF7 cells (Fig. 4a). Upon treatment with either UV-C irradiation 53 54 55 (Figs. 4c & d), docetaxel (Figs, 4e & f) or 5-FU (Figs. 4g & h), consistent and statistically 56 57 significant relationships were observed for MCF7 and T-47D cells; apoptosis was 58 59 60 characterized by a direct relationship—and viability by a corresponding inverse 61 62 11 63

64 65 relationship—with cellular GAS5 levels (Figs. 4c – 4h). Parameters of cell survival after 1 2 imatinib treatment were unrelated to cellular GAS5 levels in both cell lines (data not shown). 3 4 5 6 7 8 9 GAS5 lncRNA is sufficient to induce apoptosis in MCF7 cells 10 11 12 The transfection of breast cancer cells with GAS5 ESTs promotes apoptosis [9], but it is 13 14 15 unclear whether mature GAS5 lncRNA is sufficient to mediate this action, as all the effective 16 17 GAS5 ESTs contained retained snoRNAs. Therefore, the cell death-promoting activities of 18 19 20 mature GAS5 lncRNA (i.e., exons 1 – 12 only) and the GAS5-O1 EST (comprises snoRNA 21 22 U74, 3′ sequence of the first intron and exons 2 – 12) were compared in MCF7 cells. 23 24 25 26 27 28 29 Transfection of MCF7 cells with each GAS5 construct increased cellular GAS5 levels to a 30 31 similar extent (Fig. 5a). As expected [9], the GAS5-O1 construct increased the proportion of 32 33 34 apoptotic cells and decreased culture viability (Fig. 5b) at 24 h post-nucleofection; the GAS5- 35 36 AE construct, which encodes mature lncRNA, produced a similar quantitative response (Fig. 37 38 5b). Likewise, GAS5-O1 and GAS5-AE constructs stimulated UV-C-induced apoptosis and 39 40 41 loss of culture viability (Fig. 5c) to a similar extent. Thus, mature GAS5 lncRNA is sufficient 42 43 to promote apoptosis in MCF7 cells. 44 45 46 47 48 49 50 GAS5 lncRNA also induces apoptosis in triple-negative MDA-MB-231 cells 51 52 53 Endogenous levels of GAS5 transcripts are reduced in the TNBC cell line, MDA-MB-231, 54 55 56 relative to estrogen receptor-positive cell lines [9, 21]. To examine if TNBC cells retain 57 58 sensitivity to GAS5 lncRNA, parameters of cell survival were assessed following transfection 59 60 61 62 12 63

64 65 of MDA-MB-231 cells with the plasmid encoding mature GAS5 lncRNA. Increased GAS5 1 2 levels (Fig. 6a) were associated with increased basal apoptosis and reduced culture viability 3 4 5 (Fig. 6b) from 24 h post-nucleofection, and also enhanced the death of MDA-MB-231 cells 6 7 upon UV-C-irradiation (Fig. 6c). Thus, triple-negative MDA-MB-231 cells are sensitive to 8 9 10 GAS5 lncRNA, as for MCF7 cells. 11 12 13 14 15 16 mTOR inhibition modulates GAS5 expression in breast cancer cell lines 17 18 19 20 Clinically, the use of mTOR inhibitors may offer a simple method to enhance cellular GAS5 21 22 levels in order to promote the death of breast cancer cells. Therefore, we initially examined 23 24 the effects of rapamycin and related first generation mTOR inhibitors on breast cancer cell 25 26 27 lines. These agents inhibited the growth of all cell lines by between ca. 20 – 50%; the extent 28 29 of growth inhibition was similar for MCF7 and T-47D cells, but was noticeably smaller for 30 31 32 MDA-MB-231 cells, irrespective of the applied rapalogue (Fig. 7a). Cellular GAS5 levels 33 34 were enhanced by >50% (P < 0.05; one-way ANOVA and Bonferroni’s MCT) by rapalogue 35 36 37 treatment of MCF7 and T-47D cells, whereas they were unchanged in MDA-MB-231 cells 38 39 (Fig. 7a). In view of this resistance of MDA-MB-231 cells to first generation mTOR 40 41 42 inhibitors (which act selectively on mTORC1), we next examined the effects of inhibitors 43 44 with a broader specificity, i.e. the dual mTORC1/mTORC2 inhibitor, AZD8055, and the dual 45 46 PI3K/mTOR inhibitor, BEZ235. The pattern of cell line response to AZD8055 was similar to 47 48 49 that of rapalogues, whereas BEZ235 markedly inhibited growth and robustly elevated GAS5 50 51 levels in all cell lines (Fig. 7b). 52 53 54 55 56 57 58 59 60 61 62 13 63

64 65 Consistent with this insensitivity to rapalogues and AZD8055, MDA-MB-231 cells failed to 1 2 up-regulate cellular GAS5 levels upon cell density-induced growth arrest (Fig. 7c). In 3 4 5 contrast, both MCF7 and T-47D cells were characterized by increased GAS5 expression in 6 7 stationary versus logarithmic phases of growth (Fig. 7c). Further analysis of these data 8 9 10 confirmed that, in actively growing cultures, levels of GAS5 expression were lower (P < 11 12 0.05; one-way ANOVA and Bonferroni’s MCT) in MDA-MB-231 cells relative to both 13 14 15 MCF7 and T-47D cells, as previously reported [9]. 16 17 18 19 20 21 Discussion 22 23 24 The development of targeted therapies has revolutionised the treatment of hormone receptor- 25 26 27 positive and human epidermal growth factor receptor 2 (HER2)-positive breast cancer, but 28 29 resistance to these therapies remains problematic [25, 26]. Furthermore, there currently exists 30 31 32 no approved targeted therapy for TNBC (lacks estrogen receptor, progesterone receptor and 33 34 HER2 amplification), which frequently exhibits highly malignant behaviour and resistance to 35 36 37 conventional chemotherapies [27]. The identification of novel therapeutic targets for multiple 38 39 breast cancer subtypes is therefore important, and lncRNAs may offer new opportunities in 40 41 42 this regard, given their emerging roles as regulators of fundamental biological processes [1- 43 44 5]. Indeed, GAS5 is already of particular interest in relation to breast cancer, since its 45 46 expression is down-regulated in tumour tissue [9], and patient prognosis is significantly 47 48 49 related to GAS5 transcript levels [10]. Current findings in preclinical models demonstrate 50 51 here, for the first time, that breast cancer cell responses to several apoptotic stimuli are 52 53 54 quantitatively related to cellular GAS5 levels, suggesting a potential functional basis for the 55 56 latter clinically important relationship. The cytotoxic action of certain drugs, such as 57 58 59 imatinib, is shown to be GAS5–independent, thereby suggesting a way to bypass the 60 61 62 14 63

64 65 relationship to improve patient outcomes. Importantly, a possible therapeutic role for GAS5 1 2 lncRNA in TNBC is identified and, in this regard, dual PI3K/mTOR inhibition is 3 4 5 demonstrated to be effective in increasing GAS5 levels in such cells. 6 7 8 9 10 11 Although functional studies have previously reported that GAS5 transcripts exert apoptosis- 12 13 14 promoting effects in breast cancer cells and in a range of other cell types [9, 15, 18-21], the 15 16 consequences of reduced GAS5 expression for breast cancer cell survival have not been 17 18 19 addressed. Here we demonstrate that reductions in GAS5 expression of ca. 50 - 70% in MCF7 20 21 and T-47D cells are consistently associated with attenuated cell death in response to a range 22 23 24 of apoptosis-inducing agents (UV-C irradiation, 5-FU and docetaxel). Crucially, as in recent 25 26 experiments with prostate cancer cells [20], both the extent of apoptosis induction and the 27 28 associated loss of culture viability are quantitatively related to the extent of GAS5 silencing. 29 30 31 Thus, even small reductions in endogenous GAS5 expression may adversely impact upon the 32 33 responses of breast cancer cells to certain death-inducing stimuli, including conventional 34 35 36 chemotherapeutic agents, and this resistance to therapy may explain why breast cancer 37 38 patient survival is related to tumour GAS5 levels [10]. 39 40 41 42 43 44 45 Imatinib, which provides targeted therapy through inhibition of receptor and non-receptor 46 47 tyrosine kinases (e.g., PDGF receptors, c-kit, c-Abl and Arg) in contrast, efficiently induced 48 49 50 apoptosis in both MCF7 and T-47D cells, irrespective of cellular GAS5 levels. Thus GAS5 51 52 selectively modulates the action of chemotherapeutic agents, perhaps related to their differing 53 54 55 mechanisms of engagement of the apoptotic machinery, as suggested by the additive or 56 57 synergistic nature of imatinib action with a range of conventional chemotherapeutic agents 58 59 [28-30]. From a therapeutic perspective, the use of GAS5-independent drugs may offer an 60 61 62 15 63

64 65 improved strategy for breast cancer patients with reduced GAS5 expression, albeit further 1 2 work is required to identify additional chemotherapeutic agents which act in a GAS5- 3 4 5 independent manner in breast cancer cells. 6 7 8 9 10 11 An alternative approach may be to enhance cellular GAS5 levels prior to, or concomitant 12 13 14 with, the administration of conventional chemotherapies, in order to improve cytotoxic drug 15 16 action. Current findings confirm that GAS5 lncRNA (rather than GAS5-derived snoRNA 17 18 19 sequence) is sufficient for apoptosis-promoting activity, and further demonstrate that GAS5 20 21 lncRNA is active in the TNBC cell line, MDA-MB-231, which exhibits lower endogenous 22 23 24 GAS5 levels than estrogen receptor-positive cell lines. Consequently, the proposed approach 25 26 may be beneficial in TNBC, the treatment of which remains especially challenging [27]. Such 27 28 a therapeutic goal could be achieved by a direct approach, involving, for example, gene 29 30 31 therapy with vectors encoding GAS5 lncRNA, but the clinical application of such therapies in 32 33 general, remains a long term prospect. Since a conventional pharmacological approach would 34 35 36 be preferable in the short term, we focussed here on the mTOR pathway, which is involved in 37 38 the physiological regulation of cellular GAS5 levels [11, 14]. Only the dual PI3K/mTOR 39 40 41 inhibitor, BEZ235, elevated GAS5 levels in all cell lines. 42 43 44 45 46 47 The reasons for the resistance of GAS5 transcript levels to mTOR inhibition in MDA-MB- 48 49 50 231 cells is unknown. This cell line also failed to increase GAS5 transcript levels upon cell 51 52 density-induced growth arrest, suggesting a defect in the physiological mechanism of mTOR- 53 54 55 mediated regulation of GAS5. Mutation of the 5’-TOP sequence can be excluded however, 56 57 since the sequence of GAS5 exon 1 (contains the 5’-TOP) in MDA-MB-231 cells is identical 58 59 to that of the NCBI Reference Sequence, NR_002578.2 (data not shown). The mTOR 60 61 62 16 63

64 65 inhibitors are in increasing clinical use and resistance to such agents is becoming increasingly 1 2 evident. The growth of MDA-MB-231 cells has been noted to exhibit reduced sensitivity to 3 4 5 rapalogues [31-33], as also noted in our study. Nevertheless, basal-like cell lines are not 6 7 totally unresponsive to mTOR inhibitors, since their death can be synergistically enhanced by 8 9 10 combining such agents with cisplatin [34]. A striking finding in our study, however, was that 11 12 GAS5 levels in MDA-MB-231 cells showed complete resistance to mTORC1 inhibition by 13 14 rapalogues and to dual mTORC1/mTORC2 inhibition by AZD8055. In contrast, they 15 16 17 displayed a significant increase in response to the dual PI3K/mTOR inhibitor, BEZ235, 18 19 suggesting that mTOR-independent induction of GAS5 may prove a more productive 20 21 22 approach to improving the therapy of advanced breast cancers. 23 24 25 26 27 28 Conclusions 29 30 31 32 The extent of breast cancer cell death consequent upon exposure to certain chemotherapeutic 33 34 drugs and other apoptotic stimuli is directly related to cellular GAS5 levels, implicating 35 36 37 regulation of apoptosis as the functional role that links GAS5 expression levels with patient 38 39 survival. The use of novel targeted therapies which act independently of GAS5, or 40 41 42 combination therapies comprising conventional chemotherapeutics coupled with agents to 43 44 enhance cellular GAS5 lncRNA levels, in breast tumours could prove beneficial in improving 45 46 patient outcomes. In this regard, a wide range of mTOR inhibitors may prove useful in 47 48 49 patients with receptor-positive disease, whereas treatment options may be limited to dual 50 51 PI3K/mTOR inhibitors in TNBC patients. Findings here in breast cancer cell lines may be 52 53 54 relevant to the therapy of a wide range of other cancers, including head and neck squamous 55 56 cell carcinoma [10], glioblastoma multiforme [35], renal clear cell carcinoma [36], bladder 57 58 59 60 61 62 17 63

64 65 cancer [37], pancreatic cancer [38] and non-small-cell lung cancer [39], all of which are 1 2 characterized by deficient GAS5 expression. 3 4 5 6 7 8 9 Acknowledgements 10 11 12 This work was supported by a grant from the Breast Cancer Campaign, which is gratefully 13 14 15 acknowledged. 16 17 18 19 20 21 Conflicts of interest 22 23 24 25 The authors declare that they have no conflicts of interest. 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 18 63

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64 65 27. Engebraaten O, Vollan HK, Børresen-Dale AL (2013) Triple-negative breast cancer and 1 2 the need for new therapeutic targets. Am J Pathol 183:1064-1074 3 4 5 6 28. Kano Y, Akutsu M, Tsunoda S, Mano H, Sato Y, Honma Y, Furukawa Y (2001) In vitro 7 8 cytotoxic effects of a tyrosine kinase inhibitor STI571 in combination with commonly used 9 10 11 antileukemic agents. Blood 97:1999-2007 12 13 14 29. Sims JT, Ganguly S, Fiore LS, Holler CJ, Park ES, Plattner R (2009) STI571 sensitizes 15 16 breast cancer cells to 5-fluorouracil, cisplatin and camptothecin in a cell type-specific 17 18 19 manner. Biochem Pharmacol 78:249-260 20 21 22 30. Weigel MT, Dahmke L, Schem C, Bauerschlag DO, Weber K, Niehoff P, Bauer M, 23 24 Strauss A, Jonat W, Maass N, Mundhenke C (2010) In vitro effects of imatinib mesylate on 25 26 27 radiosensitivity and chemosensitivity of breast cancer cells. BMC Cancer 10:412 28 29 30 31. Chen Y, Zheng Y, Foster DA (2003) Phospholipase D confers rapamycin resistance in 31 32 33 human breast cancer cells. Oncogene 22:3937-3942 34 35 36 32. Chen G, Yang N, Wang X, Zheng SY, Chen Y, Tong LJ, Li YX, Meng LH, Ding J 37 38 (2010) Identification of p27/KIP1 expression level as a candidate biomarker of response to 39 40 41 rapalogs therapy in human cancer. J Mol Med (Berl) 88:941-952 42 43 44 33. Noh WC, Mondesire WH, Peng J, Jian W, Zhang H, Dong J, Mills GB, Hung MC, Meric- 45 46 47 Bernstam F (2004) Determinants of rapamycin sensitivity in breast cancer cells. Clin Cancer 48 49 Res 10:1013-1023 50 51 52 34. Wong SW, Tiong KH, Kong WY, Yue YC, Chua CH, Lim JY, Lee CY, Quah SI, Fow C, 53 54 55 Chung C, So I, Tan BS, Choo HL, Rosli R, Cheong SK, Leong CO (2011) Rapamycin 56 57 synergizes cisplatin sensitivity in basal-like breast cancer cells through up-regulation of p73. 58 59 60 Breast Cancer Res Treat 128:301-313 61 62 22 63

64 65 35. Lee J, Kotliarova S, Kotliarov Y, Li A, Su Q, Donin NM, Pastorino S, Purow BW, 1 2 Christopher N, Zhang W, Park JK, Fine HA (2006) Tumor stem cells derived from 3 4 5 glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of 6 7 primary tumors than do serum-cultured cell lines. Cancer Cell 9:391-403 8 9 10 11 36. Qiao HP, Gao WS, Huo JX, Yang ZS (2013) Long non-coding RNA GAS5 functions as a 12 13 tumor suppressor in renal cell carcinoma. Asian Pac J Cancer Prev 14:1077-1082 14 15 16 37. Liu Z, Wang W, Jiang J, Bao E, Xu D, Zeng Y, Tao L, Qiu J (2013) Downregulation of 17 18 19 GAS5 promotes bladder cancer cell proliferation, partly by regulating CDK6. PLoS One 20 21 8:e73991 22 23 24 38. Lu X, Fang Y, Wang Z, Xie J, Zhan Q, Deng X, Chen H, Jin J, Peng C, Li H, Shen B 25 26 27 (2013) Downregulation of gas5 increases pancreatic cancer cell proliferation by regulating 28 29 CDK6. Cell Tissue Res 354:891-896 30 31 32 33 39. Shi X, Sun M, Liu H, Yao Y, Kong R, Chen F, Song Y (2013) A critical role for the long 34 35 non-coding RNA GAS5 in proliferation and apoptosis in non-small-cell lung cancer. Mol 36 37 38 Carcinog. doi: 10.1002/mc.22120 [Epub ahead of print]. 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 23 63

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1 2 3 Figure legends 4 5 6 7 Fig. 1 Effect of GAS5 siRNA on basal survival and UV-C-induced cell death in MCF7 cells. 8 9 MCF7 cells (n = 5 cultures) were transfected with the indicated GAS5 siRNA or negative 10 11 control (NC) siRNA and harvested at 72 h post-transfection. Cells were treated ± UV-C 12 13 14 irradiation and replated for assessment of cell survival after a further 48 h. RT-qPCR analysis 15 16 confirmed decreased cellular GAS5 levels at 72 h post-transfection in cells treated with 17 18 19 GAS5 siRNAs (a). Silencing of GAS5 has no statistically significant effect on basal apoptosis 20 21 in MCF7 cells (b), whereas it attenuates UV-C-induced apoptosis (c). Correspondingly, 22 23 24 viability is not significantly affected in control cells (d), but it is enhanced in UV-C-treated 25 26 cells (e). Viable cell numbers are not significantly affected in both control (f) and UV-C- 27 28 treated (g) cells. Clonogenic activity is also unaffected in control cells (h) but it is enhanced 29 30 31 in UV-C-treated cells (i). *P < 0.05 & **P < 0.01 versus cells transfected with NC siRNA 32 33 (one-way ANOVA and Dunnett’s MCT) 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 24 63

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1 2 3 4 5 6 7 Fig. 2 Effect of GAS5 siRNA on basal survival and UV-C-induced cell death in T-47D cells. 8 9 T-47D cells (n = 4 cultures) were transfected with the indicated GAS5 siRNA or negative 10 11 control (NC) siRNA and harvested at 72 h post-transfection. Cells were treated ± UV-C 12 13 14 irradiation and replated for assessment of cell survival after a further 48 h. RT-qPCR analysis 15 16 confirmed decreased cellular GAS5 levels at 72 h post-transfection in cells treated with 17 18 19 GAS5 siRNAs (a). Silencing of GAS5 reduces both basal (b) and UV-C-induced apoptosis 20 21 (c). Correspondingly, viability is enhanced in control (d) and UV-C-treated cells (e). Viable 22 23 24 cell numbers are not significantly affected in both control (f) and UV-C-treated (g) cells. 25 26 Clonogenic activity is unaffected in control cells (h) but it is enhanced in UV-C-treated cells 27 28 (i). *P < 0.05, **P < 0.01 & ***P < 0.001 versus cells transfected with NC siRNA (one-way 29 30 31 ANOVA and Dunnett’s MCT) 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 25 63

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1 2 3 Fig. 3 Effect of GAS5 silencing on chemotherapeutic drug-induced death of breast cancer cell 4 5 lines. MCF7 and T-47D cells (n = 5 cultures) were transfected with the indicated GAS5 6 7 8 siRNA or negative control (NC) siRNA and, after 72 h, exposed to chemotherapeutic agents 9 10 or vehicle for 48 h. For all figure parts, apoptosis is displayed in the left-hand panel and 11 12 13 culture viability in the right-hand panel. For vehicle controls, GAS5 silencing had negligible 14 15 effects overall on apoptosis and culture viability in MCF7 cells (a), whereas it reduced 16 17 18 apoptosis and increased culture viability in T-47D cells (b). For docetaxel treatment, prior 19 20 GAS5 silencing resulted in consistent and corresponding effects on apoptosis and culture 21 22 23 viability in both MCF7 (c) and T-47D cells (d). Similar effects were observed for 5-FU 24 25 treatment of MCF7 (f) and T-47D (g) cells, whereas induction of cell death by imatinib was 26 27 unaffected by GAS5 silencing in both cell lines (h and i, respectively). *P < 0.05 & **P < 28 29 30 0.01 versus cells transfected with NC siRNA (one-way ANOVA and Dunnett’s MCT) 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 26 63

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1 2 3 Fig. 4 Parameters of cell survival are related to cellular GAS5 levels in breast cancer cell 4 5 lines. Regression analysis was performed for data from Figs. 1 – 3 against cellular GAS5 6 7 8 levels normalized relative to control (NC siRNA) values; for all figure parts, apoptosis is 9 10 displayed in the left-hand panel and culture viability in the right-hand panel. In the absence of 11 12 13 any exogenous apoptotic stimulus, apoptosis and culture viability were unrelated to GAS5 14 15 levels in MCF7 cells (a), whereas apoptosis was directly related to—and culture viability was 16 17 18 inversely related to—cellular GAS5 levels in T-47D cells (b). Apoptosis and culture viability 19 20 were also related to GAS5 levels in both MCF7 and T-47D cells upon exposure to the 21 22 23 apoptotic stimuli: UV-C irradiation (c & d); docetaxel (e & f); and 5-FU (g & h) 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 27 63

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1 2 3 Fig. 5 Comparison of the effects of GAS5 ncRNA constructs on the survival of MCF7 cells, 4 5 6 both basally and after UV-C-induced apoptosis. Cells (n = 4) were transfected with GAS5 7 8 constructs corresponding to mature lncRNA (exons 1 – 12; AE), the GAS5.O1 EST (snoRNA 9 10 11 U74 plus exons 2 – 12; O1), or empty pcDNA3 vector (V) as control. After 24 h, cells were 12 13 treated ± UV-C irradiation then re-sampled after a further 48 h. a) Cellular GAS5 levels at 24 14 15 h post-transfection are increased to a similar extent by GAS5 O1 and AE constructs. b) At 24 16 17 18 h post-transfection, the two GAS5 constructs result in similar increases in basal apoptosis 19 20 (left-hand panel) and similar decreases in basal culture viability (right-hand panel). c) The 21 22 23 two GAS5 constructs exhibit similar potency in enhancing both UV-C-induced apoptosis 24 25 (left-hand panel) and the associated loss in culture viability (right-hand panel). *P < 0.05 & 26 27 28 ***P < 0.001 versus cells transfected with pcDNA3 alone (one-way ANOVA and 29 30 Bonferroni’s MCT) 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 28 63

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1 2 3 Fig. 6 GAS5 lncRNA is pro-apoptotic in MDA-MB-231 cells. Cells (n = 4) were transfected 4 5 6 with a construct encoding mature lncRNA (AE) or empty pcDNA3 vector (V) as control and, 7 8 after 24 h, cells were treated ± UV-C irradiation, then re-sampled after a further 48 h. a) 9 10 11 Cellular GAS5 levels at 24 h post-transfection are increased by the GAS5 AE construct. b) 12 13 GAS5 lncRNA increases basal apoptosis (left-hand panel) and reduces culture viability 14 15 (right-hand panel) at 24 h post-transfection. c) GAS5 lncRNA increases UV-C-induced 16 17 18 apoptosis (left-hand panel) and the associated loss of culture viability. *P < 0.05 & ***P < 19 20 0.001 versus cells transfected with pcDNA3 alone (Student’s t-test [a & b] or one-way 21 22 23 ANOVA and Bonferrroni’s MCT [c]) 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 29 63

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1 2 3 Fig. 7 Effects of pharmacological and physiological inhibitors of mTOR activity in breast 4 5 6 cancer cell lines. a) Effect of rapamycin (1 µM; R), everolimus (10 nM; E) and temsirolimus 7 8 (10 nM; T) on culture growth (at 70 h; left-hand panel) and GAS5 levels (at 20 h; right-hand 9 10 11 panel). Data are expressed relative to the vehicle control; *P < 0.05 and **P < 0.01 versus 12 13 MDA-MB-231 cells (one-way ANOVA and Bonferrroni’s MCT; n = 4 cultures). b) Effect of 14 15 AZD8055 (50 nM) and BEZ235 (100 nM) on culture growth (at 70 h; left-hand panel) and 16 17 18 GAS5 levels (at 20 h; right-hand panel). Data are expressed relative to the vehicle control; 19 20 **P < 0.01 and ***P < 0.001 versus MDA-MB-231 cells and ++P < 0.01 versus MCF7 cells 21 22 23 (one-way ANOVA and Bonferrroni’s MCT; n = 4 cultures). c) Effect of cell density-induced 24 25 growth arrest on cellular GAS5 levels; cells were harvested at logarithmic (log) and 26 27 28 stationary (stat) phases of growth; **P < 0.01 and ***P < 0.001 versus logarithmic phase 29 30 cultures (one-way ANOVA and Bonferrroni’s MCT; n = 4 cultures) 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 30 63

64 65 Figure Click here to download high resolution image Figure Click here to download high resolution image Figure Click here to download high resolution image Figure Click here to download high resolution image Figure Click here to download high resolution image Figure Click here to download high resolution image Figure Click here to download high resolution image