SOX2OT Long Noncoding RNA Is Regulated by the UPR in Oestrogen Receptor-Positive Breast Cancer

Article SOX2OT Long Noncoding RNA Is Regulated by the UPR in Oestrogen Receptor-Positive Breast Cancer

Carole Ferraro-Peyret 1,2,3,*, Marjan E. Askarian-Amiri 1,4 , Debina Sarkar 1,4,5 , Wayne R. Joseph 1, Herah Hansji 1,4,6, Bruce C. Baguley 1 and Euphemia Y. Leung 1,4,*

1 Auckland Cancer Society Research Centre, University of Auckland, 85 Park Rd, Grafton, Auckland 1023, New Zealand; [email protected] (M.E.A.-A.); [email protected] (D.S.); [email protected] (W.R.J.); [email protected] (H.H.); [email protected] (B.C.B.) 2 Univ Lyon, ISPB Faculty of Pharmacy, INSERM 1052, CNRS5286, Cancer Research Centre of Lyon, F-69008 Lyon, France 3 Hospices Civils de Lyon, Biopathology of Tumours, GHE Hospital, F-69500 Bron, France 4 Molecular Medicine and Pathology Department, University of Auckland, 85 Park Rd, Grafton, Auckland 1023, New Zealand 5 Biochemistry Department, University of Otago, 710 Cumberland Street, Dunedin North, Dunedin 9016, New Zealand 6 Murdoch Children’s Research Institute, The Royal Children’s Hospital, Flemington Rd, Parkville, VIC 3052, Australia * Correspondence: [email protected] (C.F.-P.); [email protected] (E.Y.L.)

Abstract: Endoplasmic reticulum (ENR) stress perturbs cell homeostasis and induces the unfolded protein response (UPR). In breast cancer, this process is activated by oestrogen deprivation and is Citation: Ferraro-Peyret, C.; associated with tamoxifen resistance. We present evidence that the transcription factor SOX2 and Askarian-Amiri, M.E.; Sarkar, D.; the long noncoding RNA SOX2 overlapping transcript (SOX2OT) are upregulated in oestrogen Joseph, W.R.; Hansji, H.; Baguley, receptor-positive (ER+) breast cancer and in response to oestrogen deprivation. We examined the B.C.; Leung, E.Y. SOX2OT Long effect of the UPR on SOX2 and SOX2OT expression and the effect of SOX2OT on UPR pathways in Noncoding RNA Is Regulated by the breast cancer cell lines. The induction of the UPR by thapsigargin or glucose deprivation upregulates UPR in Oestrogen Receptor-Positive Breast Cancer. Sci 2021, 3, 26. SOX2OT expression. This upregulation is also shown with the anti-oestrogen 4OH-tamoxifen and https://doi.org/10.3390/sci3020026 mTOR inhibitor everolimus in ER + breast cancer cells that are sensitive to oestrogen deprivation or everolimus treatment. SOX2OT overexpression decreased BiP and PERK expression. This effect of Academic Editors: Aamir Ahmad and SOX2OT overexpression was conﬁrmed on BiP and PERK pathway by q-PCR. Our results show that Tomoharu Sugie a long noncoding RNA regulates the UPR and evince a new function of SOX2OT as a participant of ENR stress reprogramming of breast cancer cells. Received: 6 March 2020 Accepted: 20 March 2020 Keywords: breast cancer; LncRNA; SOX2OT; SOX2; UPR; 4-OH tamoxifen Published: 26 May 2021

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional afﬁl- Despite extensive studies in breast cancer and more detailed knowledge of its molec- iations. ular pathways, many aspects of breast cancer cell regulation are still enigmatic. Recent genomic and transcriptomic analyses showed that most of the transcripts are long noncoding RNAs (lncRNAs), and evidence for regulatory roles for lncRNAs continues to rise. The dysregulation of several lncRNAs in breast cancer has been reported [1–3] and the Copyright: © 2021 by the authors. functions of this class of transcript require elucidation. Within an intronic region of the Licensee MDPI, Basel, Switzerland. gene specifying the SOX2 overlapping transcript (SOX2OT) lncRNA lies the SOX2 gene, This article is an open access article one of the main regulators of pluripotency (Figure1,[ 4]). As described previously [4], distributed under the terms and SOX2 and SOX2OT are differentially expressed in ER+ and ER- breast cancer. They are conditions of the Creative Commons Attribution (CC BY) license (https:// both upregulated in suspension culture under conditions that prioritise spheroid forma- creativecommons.org/licenses/by/ tion. Hence, we suggest that in breast cancer, SOX2OT is key to the regulation of SOX2 4.0/). expression. However, the mechanism of action of SOX2OT in breast cancer remains to be

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fully defined. Expression analysis of murine Sox2 and Sox2OT in different developmental systemsfully deﬁned. has also Expression elucidated analysis the dynamically of murine Sox2 changingand Sox2OT expressionin different patterns developmental of these two RNAsystems species has alsoand elucidatedhas suggested the dynamicallyimportant roles changing for these expression genes in patternsnormal development of these two [5].RNA species and has suggested important roles for these genes in normal development [5].

Figure 1. Schematic of the SOX2 and SOXOT genes. The SOX2 gene lies in an intronic region ofof the theSOX2 SOX2 Overlapping Overlapping Transcript Transcriptgene gene (SOX2OT (SOX2OT). The). directionThe direction of the of transcription the transcription is shown is shown with arrows. with arrows.The triangles The triangles above each above gene each show gene the show forward the (forwardF) and reverse (F) and (R reverse) primers (R) used primers in PCR. used The in PCR.SOX2 Thegene SOX2 is enlarged. gene is enlarged.

The dysregulation of SOX2OT and SOX2 expression has also been shown in cancers such asas gliomaglioma and and kidney kidney carcinoma carcinoma [5 ],[5], and and significant significant correlations correlations in expression in expression of these of thesetwo genes two weregenes found were infound breast in cancer breast [ 4cancer], as well [4], as as in well oesophageal as in oesophageal [6] and lung [6] squamous and lung squamouscell carcinoma cell carcinoma [7–9]. We [7–9]. have We previously have previously shown in shown breast in cancer breast that cancer the that differential the dif- ferentialexpression expression of SOX2 ofand SOX2SOX2OT and SOX2OTis oestrogen is oestrogen receptor receptor dependent. dependent.SOX2OT SOX2OTand SOX2 and SOX2are more are highlymore highly expressed expressed in oestrogen in oestrogen receptor-positive receptor-positive (ER+) (ER+) thanin than ER-negative in ER-negative (ER-) (ER-)breast breast cancer cancer cell lines cell [4 lines]. Interestingly, [4]. Interestingly, high expression high expression levels of SOX2OTlevels of andSOX2OTSOX2 andare SOX2associated are associated with the sensitivitywith the sensitivity of breast of cancer breast cells cancer to tamoxifen cells to tamoxifen [4]. To investigate[4]. To investi- the gatemechanism the mechanism of action of of actionSOX2OT of SOX2OTin breast cancerin breast further, cancer we further, have examined we have theexamined role of the roleunfolded of the protein unfolded response protein (UPR) response pathway (UPR) in pathway the expression in the expression of this lncRNA. of this lncRNA. Multiple mechanisms have been described fo forr tamoxifen tamoxifen resistance resistance to to breast breast cancer. cancer. One of those pathways is known known as as the the unfold unfoldeded protein protein response response (UPR). (UPR). Three Three endoplas- endoplas- mic reticulum (ENR) stress transducers definingdefining three distinct axes of the UPR have been identifiedidentified soso farfar andand characterised characterised as as components components of of the the UPR UPR activation activation pathway pathway(Figure (Figure2) . 2).IRE1, IRE1, PERK, PERK, and and ATF6 ATF6 are are the the three three transmembrane transmembrane inducers inducers of of ENR ENR stress stress [[10].10]. TheseThese three regulators of UPR are controlled by the ENR chaperone BiP, constitutively bound to three regulators of UPR are controlled by the ENR chaperone BiP, constitutively bound to them but dissociated under ENR stress. The UPR transiently inhibits protein synthesis and them but dissociated under ENR stress. The UPR transiently inhibits protein synthesis induces the production of chaperone molecules in order to restore ENR homeostasis and and induces the production of chaperone molecules in order to restore ENR homeostasis promote cell survival [11]. The failure of this rescue mechanism results in apoptotic cell and promote cell survival [11]. The failure of this rescue mechanism results in apoptotic death [12]. cell death [12]. UPR activation is associated with poor prognosis in breast cancer [13,14]. Breast cancer cells of all subtypes have elevated UPR signalling with elevated BiP expression [15,16]. The UPR may favour oestrogen-dependent breast cancer survival when oestrogen availability is low [17–19]. The IRE-1 pathway is activated in ER+ breast cancer: it induces the splicing of XBP1 mRNA, consequently increasing the abundance of its pro-survival target XBP1s, which are strongly correlated with ER alpha expression in breast cancer [14,20]. It has been shown that silencing of XBP1, or inhibition of IRE1 by the pharmacological inhibitor STF- 80310 or MKC866, reverses resistance to anti-oestrogen therapies [21–23]. XBP1s expression promotes the survival of triple-negative breast cancer (TNBC) cells [24]. Finally, the PERK pathway is activated in breast cancer cells resistant to tamoxifen [20,21,25]. The ability of the UPR to regulate gene expression and protein synthesis and its contribution to oncogenesis have been well documented. Recent results have shown that the UPR can regulate post-transcriptional networks either by regulated IRE1-dependent decay (RIDD) of selected mRNAs [26] or by modulation of expression of micro-RNAs [27]. The

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UPR can also suppress the expression of several miRNAs that regulate either the PERK or the IRE1 pathway. However, an added layer of regulatory complexity that entails the role of lncRNA in the UPR pathway has been barely explored. Here, we investigate the gene expression pattern of SOX2OT in a UPR-induced system. Our results show that Sci 2021, 3, x FOR PEER REVIEW 3 of 13 SOX2OT expression can be upregulated by ENR stress inducers and that this lncRNA can also downregulate the PERK pathway and BiP expression in ER+ breast cancer cell lines.

FigureFigure 2. 2. SchematicSchematic of of pathways pathways involved involved in in the the UPR. UPR.

2. MaterialUPR activation and Methods is associated with poor prognosis in breast cancer [13,14]. Breast can- cer2.1. cells Cell of Lines all subtypes have elevated UPR signalling with elevated BiP expression [15,16]. The UPRAs previouslymay favour described, oestrogen-dependent the MCF-7 and breast MDA-MB-231 cancer survival cell lines when were oestrogen purchased avail- from abilitythe ATCC, is low grown [17–19]. in alpha-MEM The IRE-1 containingpathway is 5% activated foetal calf in serum ER+ (FCS),breast penicillin/streptomycincancer: it induces the splicing(100 U/mL of XBP1 and mRNA, 100 µg/mL, consequently respectively), increasing and insulin/transferrin/selenium the abundance of its pro-survivalsupplement target XBP1s,(Roche). which Long-term are strongly oestrogen-deprived correlated with sublinesER alpha TamC3 expression and TamC6in breast were cancer generated [14,20]. byIt hasgrowing been shown the MCF-7 that silencing cells in oestrogen-deprivedof XBP1, or inhibition medium of IRE1 (phenol by the pharmacological red-free RPMI 1640 inhibitor(GIBCO) STF-80310 with 5% or charcoal-stripped MKC866, reverses foetal resist bovineance to serum) anti-oestrogen and penicillin/streptomycin therapies [21–23]. XBP1s(10 U/mL expression and 10 promotesµg/mL), the for survival 10 months, of trip [28le-negative–31]. The breast T47D cancer cell line (TNBC) was purchased cells [24]. Finally,from the the ATCC, PERK grown pathway in alpha-MEM is activated containing in breast 5%cancer foetal cells calf resistant serum (FCS) to tamoxifen and peni- [20,21,25].cillin/streptomycin (100 U/mL and 100 µg/mL). TheFor ability glucose of depletion, the UPR to cells regulate were seededgene expression in six-well and plates protein and maintainedsynthesis and 48 its h under con- tributionnormal cultureto oncogenesis conditions, have i.e.,been 25 well mM docu glucosemented. concentration, Recent results 5% have FCS, shown 5% CO that2, 21%the UPRO2. can The regulate medium post-transcrip was replacedtional with networks no glucose either DMEM by regulated (GIBCO) IRE1-dependent containing 5% decay FCS, (RIDD)penicillin/streptomycin of selected mRNAs (100 [26] U/mL or by andmodulati 100 µong/mL), of expression and insulin/transferrin/selenium of micro-RNAs [27]. The UPRsupplement can also (Roche).suppress the expression of several miRNAs that regulate either the PERK or the IRE1 pathway. However, an added layer of regulatory complexity that entails the role 2.2. Reagents of lncRNA in the UPR pathway has been barely explored. Here, we investigate the gene expressionThapsigargin pattern of was SOX2OT obtained in a fromUPR-induced Applichem system. (St. Our Louis, results MO, show USA), that Everolimus SOX2OT expressionfrom Selleck can Chemicals be upregulated (Houston, by TX,ENR USA), stress and inducers 4-OH Tamoxifen and that this is from lncRNA Sigma-Aldrich can also downregulate(St. Louis, MO, the USA). PERK Anti-PERK pathway and (No. BiP 3192) expression antibody in was ER+ purchased breast cancer from cell Cell lines. Signaling Technology (Beverly, MA, USA). Anti-BIP antibody (610978) was from BD Laboratories™ α 2.(Franklin Material Lakes, and Methods NJ, USA). Antibody against -tubulin was from Sigma-Aldrich (St. Louis, MO, USA). 2.1. Cell Lines 2.3.As Protein previously Extraction described, and Western the BlotMCF-7 Analysis and MDA-MB-231 cell lines were purchased from Asthe describedATCC, grown in detail in previouslyalpha-MEM [29 containing], breast cancer 5% cellfoetal lines calf were serum grown (FCS), to log-phase, penicillin/streptomycinwashed twice with (100 ice-cold U/mL PBS,and 100 and µg/mL, lysed in re SDSspectively), lysis buffer and according insulin/transferrin/sele- to the manufac- niumturer’s supplement protocol (Cell (Roche). Signalling Long-term Technology, oestrogen-deprived Danvers, MA, sublines USA). Protein TamC3 concentration and TamC6 werewas quantiﬁedgenerated usingby growing the bicinchoninic the MCF-7 cells acid reagentin oestrogen-deprived (Sigma). Cell lysates medium containing (phenol 20red-µg free RPMI 1640 (GIBCO) with 5% charcoal-stripped foetal bovine serum) and penicillin/streptomycin (10 U/mL and 10 µg/mL), for 10 months, [28–31]. The T47D cell line was purchased from the ATCC, grown in alpha-MEM containing 5% foetal calf serum (FCS) and penicillin/streptomycin (100 U/mL and 100 µg/mL). For glucose depletion, cells were seeded in six-well plates and maintained 48 h under normal culture conditions, i.e., 25 mM glucose concentration, 5% FCS, 5% CO2, 21% O2.

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of protein were separated by electrophoresis on a 4–10% SDS-PAGE gel (Life Technologies) and transferred to a polyvinylidenedifluoride membrane (PVDF) (Millipore, Billerica, MA USA). Blocking of nonspecific binding was achieved in a 0.1% Tween 20 Tris-buffered saline solution containing 5% w/v non-fat dry milk powder for 1 h. Membranes were incubated with primary antibodies overnight at 4 ◦C, washed, and incubated with the corresponding immunoperoxidase-conjugated secondary antibody (Santa Cruz Biotechnology) for 1 h at room temperature. Bound antibody was visualised using SuperSignal West Pico (Thermo Scientific, Waltham, MA, USA) or ECL Select (Amersham) and the chemiluminescence detection system by Fujifilm Las-3000.

2.4. Reverse Transcription, cDNA Synthesis, and Quantitative PCR and PCR As described in detail previously [4], oligo-dT and random primers were used to reverse transcribe RNA with qScript Flex cDNA kit (Dnature) according to the manufacturer’s instructions. For RT-qPCR analysis, qPCR was performed using gene-speciﬁc primers (Supplementary Materials Table S1) and Sybr Green MasterMix (Life Technologies), and expression values normalised relative to GAPDH and HPRT mRNA expression.

2.5. Ectopic Expression of SOX2 and SOX2OT This has been described in detail previously [4]. Constructs overexpressing SOX2 (NM_003106) and SOX2OT (NR_004053) and control empty plasmid (vector), Ex-NEG- M95, and EX-hLUC-M90, respectively, were purchased from GeneCopoeia. Both plasmids express SV40-mCherry-IRES-puromycin resistance, allowing detection of transfected cells. Breast cancer cells (MDA-MB-231) were chosen because of the low expression of SOX2OT and SOX2 as we have previously shown [4]. The cells were transfected with 5 µg of DNA and Lipofectamine Plus (Invitrogen) according to the manufacturer’s instructions. Three biological replicates for each construct were made, the transfected cells treated with puromycin and selected on the basis of mCherry expression by ﬂuorescence-activated cell sorting (FACS), as previously described [4]. The sorted cells were maintained in the presence of puromycin.

2.6. Statistical Analysis Results are presented as mean ± SEM. As described in detail previously [4], t-tests or Mann–Whitney rank sum tests were used for comparison between two groups. Correlation analysis was performed with Spearman’s rank correlation coefficient (R) and statistical significance (p) using SigmaPlot. p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***) were indications of statistical significance.

3. Results 3.1. Relationship between Expression of SOX2OT and ENR STRESS Stress-Inducible Genes in Breast Cancer SOX2OT and SOX2 transcripts are upregulated in tamoxifen-resistant cell lines [4,32]. The activation of the UPR in ER+ breast cancer cell lines following tamoxifen treatment has also been shown previously [33–35]. We initially analysed the genome wide-RNA transcript proﬁle of breast cancer samples from the Cancer Genome Atlas (TCGA) by RNAseq dataset (TGCA8BRCA_exp_HiSeqV2-2015-02-24-160222) including 1025 samples from breast cancer patients. Interestingly, we found different patterns of expression of UPR-inducible genes relative to SOX2OT but not SOX2 (Figure3). As shown in Table1 , the expression of XBP1 and SOX2OT were positively correlated (Spearman’s correlation coefﬁcient r = 0.355, p = 2.0 × 10−7). This analysis showed no correlation between the expression of BiP and SOX2OT while the expression of the other genes examined was negatively correlated with that of SOX2OT, with a weak correlation. However, we found no correlation of ER stress-inducible genes with SOX2 with the exception of PDIA4 (r = 0.92, p = 1.4 × 10−3). Sci 2021, 3, 26 5 of 12 Sci 2021, 3, x FOR PEER REVIEW 6 of 13

Figure 3. 3. HeatHeat map map showing showing the the expression expression of SOX2 of SOX2, SOX2OT, SOX2OT, and, UPR-related and UPR-related genes. genes.It shows It shows the expression expression of of genes genes in in the the PERK PERK pathway pathway (ATF4 (ATF4, CHOP, CHOP/ddit3/ddit3, GADD, GADD/pp1r15a/pp1r15a), ENR-associ-), ENR-associated ateddegradation degradation (BiP (BiP/hspa5/hspa5, PDIA4, PDIA4),), and and thethe IRE1 pathway pathway (XBP1 (XBP1) in) inbreast breast cancer cancer samples. samples. Data Data were from TCGA TCGA invasive invasive breast breast carc carcinomainoma (TGCA8BRCA_exp_ (TGCA8BRCA_exp_ HiSeqV2-2015-02-24-160222). HiSeqV2-2015-02-24-160222). The Thesamples samples were were classiﬁed classified based based on theiron their receptor receptor status status (oestrogen (oestrogen receptor receptor (ER), (ER), HER2, HER2, or progesteroneor pro- gesterone receptor (PR)). receptor (PR)). 3.2. SOX2OT Expression Is Upregulated in ER + Stressed Cells Table 1. Spearman correlation coefﬁcients of relative gene expression for SOX2OT and SOX2 To investigate whether lncRNA SOX2OT or SOX2 is differentially expressed by ENR compared to the expression of genes of the UPR pathways in TGCA samples (n = 1025). stress, two breast cancer cell lines (MCF-7 and T47D) were treated with the UPR inducer thapsigargin (Tg). ATF4We also studied CHOP the effect of GADD two other drugs PDIA4 used to treat XBP1 ER+ breast BiP cancer cells: the anti-oestrogen 4OH-Tam and the mTOR inhibitor everolimus, both of SOX2OT −0.195 −0.167 −0.148 −0.171 0.355 0.019 whichp value have been7.26 shown× 10−12 to induce4.95 × the10− 9UPR 2.25in breast× 10−7 cancer1.92 cell× 10 lines−9 [36]2.00 and× 10 in− 7MCF-70.508 SOX2 0.00381 0.0744 −0.0549 0.0918 0.0158 0.0311 p value 0.894 9.48 × 10−3 0.848 1.36 × 10−3 0.581 0.278

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3.2. SOX2OT Expression Is Upregulated in ER + Stressed Cells To investigate whether lncRNA SOX2OT or SOX2 is differentially expressed by ENR and T47D (Supplementary Materials Figure S1). Cells were assessed for PERK phosphor- stress, two breast cancer cell lines (MCF-7 and T47D) were treated with the UPR inducer ylation and BiP induction as markers of UPR activation using Western blot analysis (Sup- thapsigargin (Tg). We also studied the effect of two other drugs used to treat ER+ breast plementary Data S1). After 16h of treatment with Tg, a significant upregulation of cancer cells: the anti-oestrogen 4OH-Tam and the mTOR inhibitor everolimus, both of SOX2OT expression was detected in both cell lines (9.3 ± 2.26 fold in MCF-7 cells and 3.8 which have been shown to induce the UPR in breast cancer cell lines [36] and in MCF-7 and ±T47D 0.96 fold (Supplementary in T47D cells) Materials (Figure 4) Figure. This S1).induction Cells were was also assessed observed for PERK for SOX2 phosphorylation expression inand both BiP cell induction lines. Treatment as markers with of UPR 4OH-Tam activation and using everolimus Western also blot upregulated analysis (Supplementary SOX2OT in MCF-7Data S1). cells, After while 16h everolimus of treatment significantly with Tg, a significant induced upregulationSOX2OT in T47D of SOX2OT cells. Theexpression SOX2 levelwas was detected significantly in both cellupregulated lines (9.3 in± MCF-72.26 fold and in T47D MCF-7 cells cells treated and 3.8 with± 0.96 Tg or fold 4OH-Tam in T47D butcells) not (Figure with everolimus.4). This induction was also observed for SOX2 expression in both cell lines. TreatmentMCF-7 with cells 4OH-Tam were also and grown everolimus under conditio also upregulatedns of glucoseSOX2OT deprivation,in MCF-7 an cells, intrinsic while inducereverolimus of the significantly UPR. After induced 48h of cultivatioSOX2OT nin in T47D glucose-depleted cells. The SOX2 medium,level was significant significantly in- creasesupregulated in the inexpression MCF-7 and of T47DSOX2OT cells and treated SOX2 with were Tg ordetected 4OH-Tam (Figure but not4C). with everolimus.

FigureFigure 4. 4. ExpressionExpression of of SOX2OTSOX2OT andand SOX2SOX2 followingfollowing UPR UPR induction induction in in ER+ ER+ breast breast cancer cancer cell cell lines. linesMCF-7. MCF-7 and T47Dand T47D cells werecells were incubated incubated in a control in a cont mediumrol medium (Ctrl) (Ctrl) or in aor medium in a medium containing containing the ER thestress-inducing ER stress-inducing agents agents thapsigargin thapsigargin (Tg, 300 (Tg, nM), 300 4OH-Tam nM), 4OH-Tam (0.1 µM), (0.1 or µM), everolimus or everolimus (10 nM) for(10 16 h. nM)(A,B for): The 16 h. effects (A,B): of The Tg, effects 4OH-Tam, of Tg, and 4OH-Tam, everolimus and oneverolimus the expression on the ofexpressionSOX2OT ofand SOX2OTSOX2 were andmeasured SOX2 were by RT-qPCR measured and by are RT-qPCR relative and to the are housekeeping relative to the geneshousekeepingHPRT and genesGAPDH HPRT.( Cand): MCF-7 GAPDHcells were. (C cultivated): MCF-7 cells in media were containingcultivated in 25 media mM glucose containing (Ctrl) 25 or mM lacking glucose glucose (Ctrl) (Glucose or lacking dep) for glucose (Glucose dep) for 48h. The expression of SOX2OT and SOX2 was measured by RT-qPCR 48h. The expression of SOX2OT and SOX2 was measured by RT-qPCR and is relative to HPRT and and is relative to HPRT and GAPDH. Error bars represent standard deviations of at least 3 inde- GAPDH. Error bars represent standard deviations of at least 3 independent experiments. * p < 0.05, pendent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001. ** p < 0.01, *** p < 0.001.

AsMCF-7 the UPR cells is wereupregulated also grown in tamoxifen-resist under conditionsant ofcells, glucose we analysed deprivation, the effects an intrinsic of Tg, 4OH-Tam,inducer of and the everolimus UPR. After in 48h two of oestrogen- cultivationdeprived in glucose-depleted and -resistant medium, breast cancer signiﬁcant cell linesincreases derived in the from expression MCF-7 cells: of SOX2OT TamC3 andand SOX2TamC6were [28–31]. detected We (Figurehave previously4C). shown that theAs sensitivity the UPR isto upregulatedmTOR inhibition in tamoxifen-resistant of these two cell lines cells, is wedifferent: analysed TamC6 the effectscells are of highlyTg, 4OH-Tam, sensitive and to everolimus, everolimus as in twocompared oestrogen-deprived to TamC3 cells and [29–31]). -resistant Here, breast we show cancer that cell Tglines upregulated derived from the expression MCF-7 cells: of TamC3SOX2OT and in TamC6both cell [28 lines,–31]. while We have SOX2 previously is upregulated shown only in the TamC6 cell line (Figure 5). 4OH-Tam did not induce the expression of these genes in TamC6 cells, whereas in TamC3 SOX2OT expression was increased (Figure 5).

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that the sensitivity to mTOR inhibition of these two cell lines is different: TamC6 cells are Sci 2021, 3, x FOR PEER REVIEW highly sensitive to everolimus, as compared to TamC3 cells [29–31]). Here, we show8 of that 13 Tg upregulated the expression of SOX2OT in both cell lines, while SOX2 is upregulated only in the TamC6 cell line (Figure5). 4OH-Tam did not induce the expression of these genes in TamC6 cells, whereas in TamC3 SOX2OT expression was increased (Figure5). EEverolimus,verolimus, on on the the other other hand, hand, induced induced a a significant signiﬁcant increase increase of of SOX2OTSOX2OT expressionexpression in in TamC6TamC6 cells cells but but not not in in TamC3 TamC3 cells, cells, while while SOX2SOX2 waswas upregulated upregulated by by everolimus everolimus in in both both cellcell lines. lines.

Figure 5. Expression of SOX2OT and SOX2 following UPR induction in oestrogen-deprived cancer Figure 5. Expression of SOX2OT and SOX2 following UPR induction in oestrogen-deprived cancer cells. TamC3 and TamC6 were incubated in a control medium (Ctrl) or in a medium containing the cells. TamC3 and TamC6 were incubated in a control medium (Ctrl) or in a medium containing the ENRENR stress-inducing stress-inducing agents agents thapsi thapsigargingargin (Tg, 300 nM), 4OH-Tam4OH-Tam (0.1(0.1µ µM),M), or or everolimus everolimus (10 (10 nM) nM) for for16h. 16h. The The expression expression of SOX2OTof SOX2OTand andSOX2 SOX2was was measured measured by RT-qPCR by RT-qPCR and isand relative is relative to that to ofthatHPRT of HPRTand GAPDH and GAPDH. Error. Error bars represent bars represent standard standard deviations deviatio of threens of independentthree independent experiments. experiments. * p < 0.05, * p** < p0.05,< 0.01, ** p ***< 0.01,p < 0.001.*** p < 0.001.

3.3.3.3. SOX2OT, SOX2OT, but but Not Not SOX2, SOX2, Regulates Regulates BiP BiP Expression Expression and and PERK PERK Activation Activation ToTo study study further further the the role role of of the the SOX2OTSOX2OT transcripttranscript in in the the UPR UPR in in breast breast cancer, cancer, we we usedused the the MDA-MB-231 MDA-MB-231 cell cell line line,, which which has has low low expression expression of SOX2OT, of SOX2OT, andand in which in which we hadwe induced had induced ectopic ectopic expression expression of SOX2OT of SOX2OT lncRNAlncRNA [4]. The [4]. activation The activation of the UPR of the path- UPR SOX2OT SOX2 waypathway in cells in overexpressing cells overexpressing either eitherSOX2OT or SOX2or was studiedwas studied at a transcriptional at a transcriptional level, level, quantifying BiP and the PERK targets ATF4 and CHOP by RT-qPCR. When SOX2OT quantifying BiP and the PERK targets ATF4 and CHOP by RT-qPCR. When SOX2OT was was overexpressed, BiP and CHOP expression was significantly reduced, to 50% of con- overexpressed, BiP and CHOP expression was significantly reduced, to 50% of control trol values (Figure6A), while XBP1s mRNA expression was not significantly changed values (Figure 6A), while XBP1s mRNA expression was not significantly changed (Figure (Figure6B). However, in SOX2 overexpressing cells ATF4 was upregulated, while CHOP 6B). However, in SOX2 overexpressing cells ATF4 was upregulated, while CHOP and BiP and BiP showed no significant change (Figure6A). XBP1s mRNA was quantified by RT- showed no significant change (Figure 6A). XBP1s mRNA was quantified by RT-qPCR and qPCR and was not modified by SOX2 overexpression (Figure6B). We further investigated was not modified by SOX2 overexpression (Figure 6B). We further investigated whether whether SOX2OT could inhibit BiP and the PERK by Western blot analysis for BiP and SOX2OT could inhibit BiP and the PERK by Western blot analysis for BiP and PERK ex- PERK expression. Our results show that the expression of BiP and PERK decreased in pression. Our results show that the expression of BiP and PERK decreased in SOX2OT SOX2OT overexpressing cells (Figure6C). overexpressing cells (Figure 6C). A positive correlation for the expression of SOX2OT and SOX2 has been reported [4,5]. Therefore, we examined whether the downregulation of PERK and BiP was dependent on the SOX2 transcription factor or whether SOX2OT reduced the expression of these proteins independently of SOX2. In SOX2-overexpressing cells (Supplementary Materials Figure S2), when BiP, CHOP, and ATF4 were quantified at the mRNA level by RT-qPCR, ATF4 was slightly but significantly upregulated, compared to the control cells (Figure6A) (Fold change = 1.32, p < 0.001).

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1.5

1.0

Fold change change Fold 0.5

Figure 6. Effect of ectopic expression of SOX2OT or SOX2 on the UPR signalling pathway. MDA-MB- Figure 6. Effect of ectopic expression of SOX2OT or SOX2 on the UPR signalling pathway. MDA- SOX2OT SOX2 MB-231231 cells cells were were transfected transfected with with plasmids plasmids containing containing the the SOX2OTgene gene (NR_004053.3) (NR_004053.3) or the or the SOX2gene (NM_003106), gene (NM_003106), or with or their with respective their respective control control vectors. vectors. (A): Expression (A): Expression of BiP, ATF4of BiP,, and ATF4CHOP, andrelative CHOP tothat relative of HPRT to thatand of GAPDHHPRT andwas GAPDH measured was by measured RT-qPCR. by ( BRT-qPCR.): Relative (B expression): Relative ofexpres-XBP1s wassion measuredof XBP1s was by RT-qPCRmeasured relative by RT-qPCR to that relative of HPRT to andthat GAPDHof HPRT.( andC): ProteinGAPDH expression. (C): Protein levels ex- of pressionBiP and PERKlevels wereof BiP examined and PERK using were Western examined Blot using analysis. Westernα-tubulin Blot analysis. was used α-tubulin as loading was control. used asError loading bars representcontrol. Error standard bars represent deviations standard of three independentdeviations of clones.three independent * p < 0.05, *** clones.p< 0.001. * p < 0.05, ** p < 0.01, *** p< 0.001. 4. Discussion ASince positive the discovery correlation of thefor adaptivethe expression response of toSOX2OT the disruption and SOX2 of endoplasmic has been reported reticu- [4,5].lum homeostasis, Therefore, we the examined UPR has emergedwhether the as having downregulation a major role of in PERK modulating and BiP the was expression depend- entof cancer-related on the SOX2 transcription genes, notably factor through or whether transcriptional SOX2OT orreduced post-transcriptional the expressionchanges. of these proteinsHere, we independently describe a new of SOX2 layer. ofIn regulatorySOX2-overexpressing mechanisms cells of (Supplementary the UPR, affected Materials by the FigurelncRNA S2),SOX2OT when BiP. , CHOP, and ATF4 were quantified at the mRNA level by RT-qPCR, ATF4Thousands was slightly of but lncRNAs significantly have upregulated, been identiﬁed compared in cancer to the cells control [37], butcells very (Figure little 6A) is (Foldknown change about = their1.32, functionsp < 0.001). and mechanisms of action. Their localisation in varying subcellular compartments (nucleus or cytoplasm), and their ability to bind to a variety of 4.targets Discussion suggest that they should regulate gene expression and also have an effect on post- transcriptionalSince the discovery regulation of or the structural adaptive interaction. response SOX2OTto the disruptionwas ﬁrst of described endoplasmic in 2009 retic- [3], ulumand little homeostasis, is known aboutthe UPR its rolehas emerged and the regulation as having ofa major its expression. role in modulatingSOX2OT is the expressed expressionin different of cancer-related cancers, including genes, breastnotably [4, 38thro,39],ugh oesophagus transcriptional [6], and or lung post-transcriptional [7,9]. Its effect on changes.proliferation Here, depends we describe on the a cell new line layer studied. of regu Inlatory lung cancer, mechanisms cell lines of SOX2OTthe UPR, inhibitsaffected cell by thecycle lncRNA progression SOX2OT by regulating. the expression of the histone–lysine N-methyltransferase enzymeThousands EZH2 [7 ].of Conversely,lncRNAs have in breast been cancer identified cell lines, in cancerSOX2OT cellsoverexpression [37], but very reducedlittle is knownproliferation about andtheir increased functions anchorage-independent and mechanisms of action. growth Their [4 ].localisation in varying sub- Sci 2021, 3, 26 9 of 12

In this study, we have shown that two ER-stress inducers —the decrease of glucose availability or the inhibition of the endoplasmic reticulum Ca2+-ATPase by Tg— induced the expression of SOX2OT. Everolimus and 4OH-Tam, two drugs that are used to treat breast cancer and are known to induce the UPR in breast cancer cell lines [21,40], also induced an upregulation of the SOX2OT transcript, suggesting that SOX2OT and the UPR are related. This was confirmed using the MDA-MB-231 TNBC (triple-negative breast cancer) cell line that has a very low expression of SOX2OT and a strong expression of BiP. Here, we have shown that the overexpression of SOX2OT downregulates BiP expression and PERK pathway activity. SOX2 lies in an intron of the SOX2OT gene and is positively correlated with SOX2OT expression in breast cancer [4]. Therefore, SOX2OT is proposed to contribute to the transcriptional regulation of SOX2 [6,7]. We observed that when MCF-7 or T47D breast cancer cells were treated with 4OH-Tam, SOX2 expression and SOX2OT expression increased concordantly. However, SOX2 overexpression neither decreased BiP expression nor affected the PERK pathway. These results show that the effect of SOX2OT on BiP or PERK pathway (measured by ATF4 and CHOP RNA expression level) is independent of SOX2. Thus, SOX2OT has other targets yet to be identified. Furthermore, when the UPR was activated by Tg or glucose depletion, we observed that the correlation between SOX2 and SOX2OT abundance was lost, suggesting that other factors regulate SOX2 expression. This dissociation of SOX2OT and SOX2 expression is also observed in TNBCs that have downregulated SOX2OT but still expressed SOX2 [4]. Feng et al. [41] have demonstrated that in TBNC, cells that are prone to EMT have a high level of expression of BiP. This high expression may arise in part from SOX2OT downregulation and may promote the selection of more aggressive phenotypes or stem cell differentiation. Other lncRNA molecules are known to be regulated by the UPR including MALAT1 [42]. Its splicing is enhanced by PERK during infection by flavivirus [41]. These results suggest that the activation of the PERK pathway enhances the translation of specific mRNAs or miRNA as well as the expression of lncRNAs. The analysis of TCGA breast cancer samples showed a positive correlation between XBP1 and SOX2OT, notably, in HER2 negative breast cancer samples (Supplementary Materials Table S2), suggesting that the IRE1 pathway could also regulate lncRNA. Numerous studies have demonstrated that oestrogen deprivation induced a UPR [33–35]. In ER+ breast cancer cell lines, ER alpha-targeted therapy increased aggregation of ER alpha in the cytoplasm and increased UPR signalling [43]. The activation of UPR contributes to the development of resistance to oestrogen deprivation, as demonstrated by the overexpression of XBP1s [23] and BiP [44,45] or the activation of the PERK pathway [20,21,25]. Since SOX2OT is expressed early during UPR activation, it can participate in the reprogramming of gene expression by the UPR during the acute phase of oestrogen deprivation-mediated stress and lead to the emergence of resistant phenotypes. When treated with 4OH-Tam, MCF-7, T47D or TamC3 cells increased the expression of SOX2OT, whereas, in TamC6 cells, 4OH-Tam has no effect on SOX2OT expression, suggesting that in some resistant cell lines, upon chronic induction of the UPR, the mechanisms of regulation of SOX2OT expression are lost. The same observation was made following treatment with the mTOR inhibitor everolimus that has no effect on SOX2OT expression when cells are resistant to mTOR inhibition. Together, our results show that long noncoding RNA can be considered a regulator of the UPR and provide evidence for a new function of SOX2OT as a participant of ENR stress reprogramming of breast cancer cells. Whether this LncRNA mediates its function through interactions with proteins, RNA, DNA, or a combination of these remains to be investigated. Therefore, overcoming SOX2OT overexpression and its effect on the PERK pathway or on SOX2 expression could result in the adaptation of the cells to ENR stress and consequently in the resistance to anti-oestrogen treatment and/or the promotion of EMT (Figure7). Further investigations are needed to determine whether SOX2OT may provide a basis for new therapeutics to modify UPR in breast cancer. Sci 2021, 3, x FOR PEER REVIEW 11 of 13

Together, our results show that long noncoding RNA can be considered a regulator of the UPR and provide evidence for a new function of SOX2OT as a participant of ENR stress reprogramming of breast cancer cells. Whether this LncRNA mediates its function through interactions with proteins, RNA, DNA, or a combination of these remains to be investigated. Therefore, overcoming SOX2OT overexpression and its effect on the PERK pathway or on SOX2 expression could result in the adaptation of the cells to ENR stress Sci 2021, 3, 26 and consequently in the resistance to anti-oestrogen treatment and/or10 of the 12 promotion of EMT (Figure 7). Further investigations are needed to determine whether SOX2OT may provide a basis for new therapeutics to modify UPR in breast cancer.

Figure 7. HypotheticalFigure 7. Hypothetical schema of SOX2OT schemalncRNA of SOX2OT and lncRNA UPR interrelation. and UPR interrelation.

Supplementary Materials:SupplementaryThe following Materials: are The available following online atare https://www.mdpi.com/article/10 available online at www.mdpi.com/xxx/s1, .3390/sci3020026/s1Figure, Figure S1: S1: Expression Expression of of SOX2OTSOX2OTlncRNA lncRNA and andSOX2 SOX2following following UPR induction UPR induction in ER+ in ER+ breast cancerbreast cell cancer lines, cell Figure lines, S2:Figure Comparison S2: Comparison of SOX2 expressionof SOX2 expression in control andin control SOX2 and SOX2 over- overexpressing MDA-MB-231,expressing MDA-MB-231, Table S1: List ofFigure primers S1: used Figure in these S1: experiments, Table S1: List Table of S2:primers Spearman used in these experi- correlation coefficientsments of, Table relative S2: gene Spearman expression correlation for SOX2OT coefficientscompared of to relative the expression gene expression of genes for SOX2OT of the UPR pathwayscompared in TCGA to the Her2 expression neg/pos orof Hormonegenes of the neg/pos UPR pathways samples in TCGA Her2 neg/pos or Hormone neg/pos samples Author Contributions: Conceptualization, C.F.-P., E.Y.L. and M.E.A.-A.; methodology, C.F.-P., D.S. and W.R.J.; software, C.F.-P. and H.H.; validation, E.Y.L. and M.E.A.-A.; formal analysis, C.F.-P. and Author Contributions: Conceptualization, C.F.-P., E.Y.L. and M.E.A.-A.; methodology, C.F.-P., D.S. D.S.; investigation, C.F.-P.; resources, E.Y.L. and M.E.A.-A.; data curation, C.F.-P.; writing—original and W.R.J.; software, C.F.-P. and H.H.; validation, E.Y.L. and M.E.A.-A.; formal analysis, C.F.-P. and draft preparation, C.F.-P., E.Y.L., M.E.A.-A. and B.C.B.; writing—review and editing, C.F.-P., E.Y.L.; D.S.; investigation, C.F.-P.; resources, E.Y.L. and M.E.A.-A.; data curation, C.F.-P.; writing—original visualization, all authors; supervision, B.C.B.; project administration, B.C.B..; funding acquisition, C.F.- draft preparation, , C.F.-P., E.Y.L., M.E.A.-A. and B.C.B.; writing—review and editing, C.F.-P., P., E.Y.L. and M.E.A.-A.; All authors have read and agreed to the published version of the manuscript. E.Y.L.; visualization, all authors; supervision, B.C.B.; project administration, B.C.B..; funding acqui- Funding: C.F.-P. issition, supported C.F.-P., by E.Y.L. La Ligue and Sa M.E.A.-A.;ône et Loire All and authors the Hospices have read Civils and de agreed Lyon. to M.E.A.-A. the published version of is supported by the manuscript. Genesis Oncology Trust fund. E.Y.L. is supported by the NZ Breast cancer foundation. Funding: C.F.-P. is supported by La Ligue Saône et Loire and the Hospices Civils de Lyon. M.E.A.- Institutional ReviewA. is Boardsupported Statement: by the NotGenesis applicable. Oncology Trust fund. E.Y.L. is supported by the NZ Breast cancer foundation. Informed Consent Statement: Not applicable. Institutional Review Board Statement: Not applicable. Data Availability Statement: Data and materials are available on request from the corresponding authors. Informed Consent Statement: Not applicable. Acknowledgments:DataWe Availability thank G.J. FinlayStatement: for his Data careful and readingmaterials of are the available paper and on the request constructive from the corresponding comments he gave.authors. C.F.-P. is supported by La Ligue Saône et Loire and the Hospices Civils de Lyon. M.E.A.-A. is supported by the Genesis Oncology Trust. E.Y.L. is supported by the NZ Breast Acknowledgments: We thank G.J. Finlay for his careful reading of the paper and the constructive Cancer Foundation.comments he gave. C.F.-P. is supported by La Ligue Saône et Loire and the Hospices Civils de Lyon. Conflicts of Interest:M.E.A.-A.The authors is supported declare by no the conflict Genesis of interest.Oncology Trust. E.Y.L. is supported by the NZ Breast Cancer Foundation. References Conflicts of Interest: The authors declare no conflict of interest. 1. Hansji, H.; Leung, E.Y.; Baguley, B.C.; Finlay, G.J.; Askarian-Amiri, M.E. Keeping abreast with long non-coding RNAs in mammary gland development and breast cancer. Front. Genet. 2014, 5, 379. [CrossRef][PubMed] 2. Su, X.; Malouf, G.G.; Chen, Y.; Zhang, J.; Yao, H.; Valero, V.; Weinstein, J.N.; Spano, J.-P.; Meric-Bernstam, F.; Khayat, D.; et al. 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