BMI1-Mediated Pemetrexed Resistance in Non-Small Cell Lung Cancer Cells Is Associated with Increased SP1 Activation and Cancer Stemness

cancers

Article BMI1-Mediated Pemetrexed Resistance in Non-Small Cell Lung Cancer Cells Is Associated with Increased SP1 Activation and Cancer Stemness

Huan-Ting Shen 1,2, Peng-Ju Chien 3, Shih-Hong Chen 3, Gwo-Tarng Sheu 1 , Ming-Shiou Jan 1,4,5 , Bing-Yen Wang 6,7,8,9,10,11,* and Wen-Wei Chang 3,12,*

1 Institute of Medicine, Chung Shan Medical University, No.110, Sec.1, Jianguo N.Rd., Taichung City 40201, Taiwan; [email protected] (H.-T.S.); [email protected] (G.-T.S.); [email protected] (M.-S.J.) 2 Department of Pulmonary Medicine, Taichung Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, No. 88, Sec. 1, Fengxing Rd., Tanzi Dist., Taichung City 427, Taiwan 3 Department of Biomedical Sciences, Chung Shan Medical University, No.110, Sec.1, Jianguo N.Rd., Taichung City 40201, Taiwan; [email protected] (P.-J.C.); [email protected] (S.-H.C.) 4 Immunology Research Center, Chung Shan Medical University, No.110, Sec.1, Jianguo N.Rd., Taichung City 40201, Taiwan 5 Division of Allergy, Immunology and Rheumatology, Department of Internal Medicine, Chung Shan Medical University, No.110, Sec.1, Jianguo N.Rd., Taichung City 40201, Taiwan 6 Division of Thoracic Surgery, Department of Surgery, Changhua Christian Hospital, No. 135, Nanhsiao Street, Changhua County 500, Taiwan 7 School of Medicine, Chung Shan Medical University, No.110, Sec.1, Jianguo N.Rd., Taichung City 40201, Taiwan 8 School of Medicine, College of Medicine, Kaohsiung Medical University, No.100, Shin-Chuan 1st Road, Sanmin Dist., Kaohsiung City 80708, Taiwan 9 Institute of Genomics and Bioinformatics, National Chung Hsing University, No.145, Xingda Rd., South Dist., Taichung City 402, Taiwan 10 Ph.D. Program in Translational Medicine, National Chung Hsing University, No.145, Xingda Rd., South Dist., Taichung City 402, Taiwan 11 Center for General Education, Ming Dao University, No.369, Wen-Hua Rd., Pitou Township, ChangHua County 52345, Taiwan 12 Department of Medical Research, Chung Shan Medical University Hospital, No.110, Sec.1, Jianguo N.Rd., Taichung City 40201, Taiwan * Correspondence: [email protected] (B.-Y.W.); [email protected] (W.-W.C.); Tel.: +886-4-7238595 (ext. 7266) (B.-Y.W.); +886-4-24730022 (ext. 12305) (W.-W.C.); Fax: +886-4-7228289 (B.-Y.W.); +886-4-23248187 (W.-W.C.) Received: 15 June 2020; Accepted: 21 July 2020; Published: 27 July 2020

Abstract: Lung cancer is the leading cause of cancer death worldwide and the therapeutic strategies include surgery, chemotherapy and radiation therapy. Non-small cell lung cancers (NSCLCs) account for around 85% of cases of lung cancers. Pemetrexed is an antifolate agent that is currently used as the second line chemotherapy drug in the treatment of advanced NSCLC patients with a response rate of 20–40%. The search for any combination therapy to improve the efficacy of pemetrexed is required. The existence of cancer stem cells (CSCs) is considered as the main reason for drug resistance of cancers. In this study, we first found that pemetrexed-resistant NSCLC cells derived from A549 cells displayed higher CSC activity in comparison to the parental cells. The expression of CSC related proteins, such as BMI1 or CD44, and the epithelial–mesenchymal transition (EMT) signature was elevated in pemetrexed-resistant NSCLC cells. We next discovered that the overexpression of BMI1 in A549 cells caused the pemetrexed resistance and inhibition of BMI1 by a small molecule inhibitor, PTC-209, or transducing of BMI1-specific shRNAs suppressed cell growth and the expression of thymidylate synthase (TS) in pemetrexed-resistant A549 cells. We further identified that BMI1 positively regulated

Cancers 2020, 12, 2069; doi:10.3390/cancers12082069 www.mdpi.com/journal/cancers Cancers 2020, 12, 2069 2 of 16

SP1 expression and treatment of mithramycin A, a SP1 inhibitor, inhibited cell proliferation, as well as TS expression, of pemetrexed-resistant A549 cells. Furthermore, overexpression of BMI1 in A549 cells also caused the activation of EMT in and the enhancement of CSC activity. Finally, we demonstrated that pretreatment of PTC-209 in mice bearing pemetrexed-resistant A549 tumors sensitized them to pemetrexed treatment and the expression of Ki-67, BMI1, and SP1 expression in tumor tissues was observed to be reduced. In conclusion, BMI1 expression level mediates pemetrexed sensitivity of NSCLC cells and the inhibition of BMI1 will be an eﬀective strategy in NSCLC patients when pemetrexed resistance has developed.

Keywords: BMI1; SP1; pemetrexed resistance; non small cell lung cancer; cancer stem cells; epithelial–mesenchymal transition

1. Introduction Lung cancer is the leading cause of cancer-related death in the world. Non-small-cell lung cancer (NSCLC) represents 87% of patients with lung cancer, and is clinically divided into two major subtypes: adenocarcinoma and squamous cell carcinoma [1]. Most lung cancer patients present with advanced-stage disease and the current 5-year survival for lung cancer is only 18% [2]. Even in patients with early-stage resectable or locally advanced disease after standard treatment, the lung cancer recurrence rate is up to 90% [3]. Over the past two decades, multiple treatments are under clinical practice for NSCLC patients such as surgery, chemotherapy, targeted therapy, immunotherapy and radiotherapy. However, systemic chemotherapy is still the principal treatment for NSCLC, especially as palliative care for late-stage patients [4]. Platinum-based chemotherapy has been used for standard first-line regimen for advanced NSCLC since 2001 [5]. In addition to platinum, pemetrexed, one of the antifolates, has also been used as a first-line drug to treat NSCLC since 2008 [6]. The interference of folate metabolism by pemetrexed leads to ineffective DNA synthesis and eventually to failure of proliferation in tumor cells. Pemetrexed acts not only with platinum as first-line regimen but also as a single agent for second-line treatment in advanced patients. However, after long-term use of chemotherapy, NSCLC will develop chemoresistance through several mechanisms such as enhanced DNA repair systems, activated epithelial-to-mesenchymal transition (EMT), increased number of cancer stem cells (CSCs), impaired drug uptake, reinforced drug elimination, defective polyglutamylation process, higher expression of oncoprotein thyroid transcription factor-1 (TTF-1) and pemetrexed target enzymes [7]. In NSCLCs, the response rate of pemetrexed treatment is 20–40% [8]. It indicates that resistance to pemetrexed treatment exists among NSCLC patients. The existence of CSCs is considered as the main reason for the drug resistance of cancers [9–11]. The expression of stemness genes has been reported to be associated with chemoresistance in cancers [12,13]. B-cell-specific Moloney leukemia virus insertion site 1 (BMI1), a structural component of the polycomb repressive complex, is functionally associated with the self-renewal of cancer stem cells resulting in the chemo- and radiation resistance of tumors [14]. BMI1 plays an important role in the regulation of gene expression and cellular physiology, including cell cycle control, DNA damage repair, and EMT [15–17]. Inhibition of BMI1 can induce apoptosis in tumor cells and elevate the sensitivity of tumor cells to chemotherapy and radiotherapy for tongue and breast cancer [18,19]. BMI1 overexpression was found as an efficient prognostic marker for NSCLC with increased tumor size, poor differentiation, more distant metastasis, and worse survival [20]. Therefore, we designed a study to understand the signaling pathway of BMI1-induced pemetrexed resistance in NSCLC. In the present study, we firstly observed the upregulation of BMI1 in pemetrexed-resistant NSCLC cells derived from A549 cells. The overexpression of BMI1 in A549 cells caused pemetrexed resistance and knockdown of BMI1 in resistant cells, sensitized them to pemetrexed treatment, Cancers 2020, 12, 2069 3 of 16 and viceCancers versa. 2020, 12 We, x FOR also PEER demonstrated REVIEW that BMI1 could induce Specificity protein 1 (SP1) expression3 of 16 and lead to the upregulation of thymidylate synthase (TS, gene name as TYMS), a key enzyme for vice versa. We also demonstrated that BMI1 could induce Specificity protein 1 (SP1) expression and pemetrexedlead to resistance.the upregulation In addition, of thymidylate we found synthase that BMI1 (TS, induced gene name the EMT as TYMS), process a andkey enhancedenzyme for cancer stemnesspemetrexed when overexpressedresistance. In addition, in A549 we cells. found We that also BMI1 provided induced the the evidence EMT process that pre-treatment and enhanced with PTC-209,cancer a BMI1stemness inhibitor, when overexpressed could sensitize in A549 NSCLC cells. tumorsWe also derivedprovided from the evidence pemetrexed-resistant that pre-treatment cells to pemetrexedwith PTC-209, treatment. a BMI1 inhibitor, could sensitize NSCLC tumors derived from pemetrexed-resistant cells to pemetrexed treatment. 2. Results 2. Results 2.1. CSC Activity and EMT Signature Is Upregulated in Pemetrexed-Resistant NSCLC Cells 2.1. CSC Activity and EMT Signature Is Upregulated in Pemetrexed-Resistant NSCLC Cells We have established the pemetrexed-resistant NSCLC cells from A549 cells, called A400 cells [21]. Given theWe important have established role of CSCthe pemetrexed-resistant in drug resistance [NSCLC9], we firstcells comparedfrom A549 thecells, CSC called activity A400 cells between [21]. Given the important role of CSC in drug resistance [9], we first compared the CSC activity A549 and A400 cells. By tumorsphere cultivation, the CSC activity was greater in A400 cells than that between A549 and A400 cells. By tumorsphere cultivation, the CSC activity was greater in A400 cells of A549 cells (Figure1A). BMI1 [ 14], CD44 [22], or Notch1 [23] has been reported as the important than that of A549 cells (Figure 1A). BMI1 [14], CD44 [22], or Notch1 [23] has been reported as the positiveimportant regulator positive in the regulator self-renewal in the self-renewal capability capability of lung CSCs. of lung By CSCs. western By western blotanalysis, blot analysis, the the protein expressionsprotein ofexpressions BMI1, CD44, of BMI1, or activated CD44, or Notch1 activated (notch Notch1 intracellular (notch intracellular domain, NICD),domain, wereNICD), upregulated were in A400upregulated cells in comparisonin A400 cells in to comparison parental A549to pare cellsntal A549 (Figure cells1B). (Figure Aldehyde 1B). Aldehyde dehydrogenase dehydrogenase (ALDH) isoforms,(ALDH) such isoforms, as ALDH1A1 such as ALDH1A1 or ALDH1A2, or ALDH1A2, have been have reported been reported as CSC as CSC markers markers in severalin several cancer typescancer [24,25 types]. However, [24,25]. weHowever, observed we thatobserved the expression that the expression of ALDH1A1 of ALDH1A1 or ALDH1A2 or ALDH1A2 was reduced was in pemetrexedreduced A400in pemetrexed cells when A400 compared cells when to thecompared parental to A549the parental cells (Figure A549 cells1C). It(Figure was consistent1C). It was with a previousconsistent report with from a previous Okudela report K etfrom al., Okudela in which K itet wasal., in described which it was that described ALDH1A1 that mightALDH1A1 function might function as a tumor suppressor in NSCLC [26]. In addition, we also observed the elevated EMT as a tumor suppressor in NSCLC [26]. In addition, we also observed the elevated EMT signature in signature in pemetrexed-resistant A400 cells by the downregulation of E-cadherin, the epithelial pemetrexed-resistant A400 cells by the downregulation of E-cadherin, the epithelial marker, and the marker, and the upregulation of N-cadherin, the mesenhymal marker, and Snail1/ZEB1, the upregulationtranscription of N-cadherin,factors involved the mesenhymalin the EMT proce marker,ss (Figure andSnail1 1D). /TheseZEB1, data the transcriptionsuggest that the factors involvedpemetrexed in the EMT resistance process in (FigureNSCLC 1cellsD). Thesemay be data asso suggestciated with that the the increased pemetrexed CSC resistance activity and in NSCLCthe cells mayelevated be associatedEMT signature. with the increased CSC activity and the elevated EMT signature.

FigureFigure 1. Cancer 1. Cancer stem stem cell cell (CSC) (CSC) activity activity and and epithelial-to-mesenchymal epithelial-to-mesenchymal transition transition (EMT) (EMT) signature signature is upregulatedis upregulated in pemetrexed-resistant in pemetrexed-resistant non-small-cell non-small-cell lung lung cancer cancer (NSCLC) (NSCLC) cells.cells. ( (AA)) The The CSC CSC activity activity in A549in cells A549 or theircells derivedor their pemetrexed-resistantderived pemetrexed-resista A400nt cells A400 was cells determined was determined by tumorsphere by tumorsphere cultivation. Primarycultivation. tumorspheres Primary (sph1) tumorspheres were counted (sph1) atwere Day counted 21 by anat invertedDay 21 by light an inverted microscopy light aftermicroscopy cultivation. Singleafter cells cultivation. of primary Single tumorspheres cells of primary were tumorsph obtainederes by HyQTasewere obtained dissociation by HyQTase and dissociation used for secondary and tumorsphereused for secondary formation tumorsphere (sph2). ** p formation< 0.01. (B (sph2).,C) The **, totalp < 0.01. proteins (B,C) The were total collected proteins from were A549collected or A400 cells and western blotting was performed to determine the expression of cancer stemness factors (B),

aldehyde dehydrogenase (ALDH) isoforms (C), or EMT-related proteins (D). All the experiments were done two times and data from one experiment were presented. Cancers 2020, 12, x FOR PEER REVIEW 4 of 16

from A549 or A400 cells and western blotting was performed to determine the expression of cancer stemness factors (B), aldehyde dehydrogenase (ALDH) isoforms (C), or EMT-related proteins (D). All Cancersthe2020 experiments, 12, 2069 were done two times and data from one experiment were presented. 4 of 16

2.2. The Expression Level of BMI1/Sp1/Thymidylate Synthase Is Correlated with Pemetrexed Sensitivity in 2.2. The Expression Level of BMI1/Sp1/Thymidylate Synthase Is Correlated with Pemetrexed Sensitivity in NSCLCNSCLC Cells Cells WeWe also also took took another another NSCLC NSCLC cell cell line, line, H1355, H1355, to to compare compare the the pemetrexed pemetrexed sensitivity sensitivity and and the the resultsresults displayed displayed that that A549 A549 were were the the most most sensit sensitiveive NSCLC NSCLC cells cells followed followed by by H1355 H1355 and and A400 A400 cells cells (Figure(Figure 22A).A). It has been reported that the upregulationupregulation of thymidylate synthasesynthase (TS) is one of the reasonsreasons for for pemetrexed pemetrexed resistance resistance [27]. [27]. Overexpressi Overexpressionon of of BMI1 BMI1 is is also also found found in in cancer cancer cells cells with with resistanceresistance to chemotherapy agentsagents [ 14[14].]. We We next next compared compared the the expression expression of BMI1,of BMI1, SP1, SP1, or TS or in TS A549, in A549,A400, A400, or H1355 or H1355 NSCLC NSCLC cells by cells western by western blot. Allblot. these All threethese protein’sthree protein’s expressions expressions in A400 in orA400 H1355 or H1355cells were cells higher were thanhigher those than of A549those cells of A549 (Figure cells2B). Here,(Figure we 2B). hypothesize Here, we that hypothesize the upregulation that the of upregulationBMI1/SP1 may of leadBMI1/SP1 to TS overexpressionmay lead to TS overex and pemetrexedpression and resistance pemetrexed in NSCLC resistance cells. in NSCLC cells.

FigureFigure 2.2. TheThe expression expression level level of B-cell-specific of B-cell-specific Moloney leukemiaMoloney virusleukemia insertion virus site 1insertion (BMI1)/Specificity site 1 (BMI1)/Specificityprotein 1 (SP1)/thymidylate protein 1 (SP1)/ synthasethymidylate (TS) is correlatedsynthase (TS) with is pemetrexedcorrelated with sensitivity pemetrexed in NSCLC sensitivity cells. in(A NSCLC) Three cells. NSCLC (A) cells Three (A549, NSCLC A400, cells or (A549, H1355) A400, were or seeded H1355) into were a 96-well-plateseeded into a at 96-well-plate 1000 cells/well at 1000and treatedcells/well with and the treated indicated with concentrationthe indicated ofconc pemetrexed.entration of The pemetrexed. cell viability The was celldetermined viability was by determined3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) bromide reagent (MTT) at 96 h reagent after treatment. at 96 h after(B) The treatment. total proteins (B) The were total harvested proteins were from harvested three NSCLC from cells three and NSCLC the expression cells and ofthe BMI1 expression or TS was of BMI1determined or TS was by western determined blot. Allby thewestern experiments blot. All were the doneexperiments three times were and done data three from times one experiment and data fromwere one presented. experiment were presented. 2.3. Manipulation of BMI1 Expression Level in NSCLC Cells Changes the Pemetrexed Sensitivity 2.3. Manipulation of BMI1 Expression Level in NSCLC Cells Changes the Pemetrexed Sensitivity We next examined if overexpression of BMI1 in A549 cells could induce pemetrexed resistance. We next examined if overexpression of BMI1 in A549 cells could induce pemetrexed resistance. From Figure3A, the overexpression of BMI1 in A549 cells induced pemetrexed resistance in comparison From Figure 3A, the overexpression of BMI1 in A549 cells induced pemetrexed resistance in to control cells (Figure3A). We also found that Sp1 expression was upregulated in BMI1-overexpressed comparison to control cells (Figure 3A). We also found that Sp1 expression was upregulated in BMI1- A549 cells (Figure3B). To further investigate the e ffects of BMI1 inhibition in pemetrexed-resistant overexpressed A549 cells (Figure 3B). To further investigate the effects of BMI1 inhibition in A400 cells, the knockdown of BMI1 in A400 cells was performed by lentiviral delivery of specific pemetrexed-resistant A400 cells, the knockdown of BMI1 in A400 cells was performed by lentiviral shRNAs (Figure3C). The decreased cell growth of A400 cells was observed after knockdown of BMI1 delivery of specific shRNAs (Figure 3C). The decreased cell growth of A400 cells was observed after with or without pemetrexed treatment (Figure3D). Given that the sensitivity of pemetrexed in NSCLC knockdown of BMI1 with or without pemetrexed treatment (Figure 3D). Given that the sensitivity of cells was thought to be associated with the level of TS expression [28], we next checked the expression pemetrexed in NSCLC cells was thought to be associated with the level of TS expression [28], we next of TS in NSCLC cells after inhibition of BMI1 protein expression or its bioactivity. The treatment of checked the expression of TS in NSCLC cells after inhibition of BMI1 protein expression or its a BMI1 inhibitor, PTC-209, caused the down-regulation of TS in both A400 and H1355 cells (Figure4A). bioactivity. The treatment of a BMI1 inhibitor, PTC-209, caused the down-regulation of TS in both We also found that the treatment of PTC-209 in pemetrexed A400 cells caused the cell cycle arrest at the A400 and H1355 cells (Figure 4A). We also found that the treatment of PTC-209 in pemetrexed A400 G1 phase in a dose-dependent manner (Figure4B). The knockdown of BMI1 by specific shRNAs also cells caused the cell cycle arrest at the G1 phase in a dose-dependent manner (Figure 4B). The suppressed TS expression in both A400 and H1355 cells (Figure4C). From these results, it suggests that

BMI1 expression level determines pemetrexed sensitivity in NSCLC cells. Cancers 2020, 12, x FOR PEER REVIEW 5 of 16 knockdown of BMI1 by specific shRNAs also suppressed TS expression in both A400 and H1355 cells

(FigureCancers 2020 4C)., 12 ,From 2069 these results, it suggests that BMI1 expression level determines pemetrexed5 of 16 sensitivity in NSCLC cells.

FigureFigure 3. 3. ManipulatingManipulating BMI1 BMI1 expression expression in NSCLC in NSCLC cells cellsinfluences influences the pemetrexed the pemetrexed sensitivity. sensitivity. (A,B) A549(A,B) cells A549 were cells transduced were transduced with lentiviruses with lentiviruses of tRPF of control tRPF control(A549-tRFP) (A549-tRFP) or flag-tagged-BMI1 or flag-tagged-BMI1 (A549- BS1)(A549-BS1) and selected and selected with 20 withµg/mL 20 µblasticiding/mL blasticidin S for 96 Sh. for The 96 expressions h. The expressions of flag tagged-BMI1 of flag tagged-BMI1 and TS inand A549-tRFP TS in A549-tRFP or A549-BS1 or A549-BS1 were examined were examined by western by blot western (A). The blot pemetrexed (A). The pemetrexed sensitivity sensitivity of A549- tRFPof A549-tRFP or A549-BS1 or A549-BS1 cells were cells determined were determined by MTT by assay MTT (B assay). *, p ( B<). 0.05. * p < (0.05.C,D) A400 (C,D) cells A400 were cells transducedwere transduced with withlentiviruses lentiviruses carrying carrying sh-LacZ sh-LacZ or sh-BMI1 or sh-BMI1 (#1 (#1or or#2) #2) and and selected selected with with 2 2µg/mLµg/mL puromycinpuromycin forfor 4848 h. h. The The survived survived cells cells were were harvested harvested and BMI1and BMI1 expression expression was examined was examined by western by westernblot (C) orblot pemetrexed (C) or pemetrexed sensitivity sensitivity was examined was examined by MTT assay by MTT (D). *assayp < 0.05. (D). All*, p the < 0.05. experiments All the experimentswere done three were times done and three data times from and one da experimentta from one were experiment presented. were presented.

Cancers 2020, 12, 2069 6 of 16 Cancers 2020, 12, x FOR PEER REVIEW 6 of 16

FigureFigure 4. 4. InhibitionInhibition of ofBMI1 BMI1 in NSCLC in NSCLC cells cells down-regulates down-regulates TS expression. TS expression. (A) BMI1 (A) inhibitor, BMI1 inhibitor, PTC- 209,PTC-209, was used was to used treat to A400 treat or A400 H1355 or H1355 NSCLC NSCLC cells at cells the atindicted the indicted concentratio concentrationn for 48 forh and 48 hthe and total the proteinstotal proteins were harvested were harvested for determination for determination of BMI1 of BMI1or TS orexpression TS expression by western by western blot. ( blot.B) A400 (B) A400cells werecells treated were treated with the with indicated the indicated concentration concentration of PTC-209 of PTC-209 for 24 h forand 24 harvested h and harvested for cell cycle for cellanalysis cycle usinganalysis propidium using propidium iodide (PI) iodide staining (PI) stainingand the andfluorescence the fluorescence was detected was detected by a flow by acytometry. flow cytometry. As a vehicleAs a vehicle control,control, 0.1% dimethyl 0.1% dimethyl sulfoxide sulfoxide (DMSO) (DMSO) was used. was used.The percentage The percentage of cells of at cells each at cell each cycle cell phasecycle phasewas quantified was quantified by FlowJo by FlowJo software. software. (C) A400 (C) or A400 H1355 or H1355NSCLC NSCLC cells were cells transduced were transduced with lentiviruseswith lentiviruses carrying carrying sh-LacZ sh-LacZ or sh-BMI1 or sh-BMI1 (#1 or (#1#2) for or #2)48 h for and 48 the h and protein the protein expression expression of BMI1 of or BMI1 TS wasor TS determined was determined by western by western blot. blot.All the All experiments the experiments were were done done two two times times and and data data from from one one experimentexperiment were were presented. presented. 2.4. BMI1 Is the Upstream Signaling Molecule of Sp1 Protein Expression in Pemetrexed-Resistant 2.4. BMI1 Is the Upstream Signaling Molecule of Sp1 Protein Expression in Pemetrexed-Resistant NSCLC NSCLC Cells Cells It has been reported that SP1 could trans-activate TS expression in MCF7 breast cancer cells [29]. It has been reported that SP1 could trans-activate TS expression in MCF7 breast cancer cells [29]. We next examined the role of SP1 in pemetrexed NSCLC cells. The protein expression of Sp1 was more We next examined the role of SP1 in pemetrexed NSCLC cells. The protein expression of Sp1 was upregulated in A400 cells than that of parental A549 cells (Figure5A). Overexpression of BMI1 in A549 more upregulated in A400 cells than that of parental A549 cells (Figure 5A). Overexpression of BMI1 cells upregulated both SP1 and TS expression (Figure5B). The SP1 inhibitor, mithramycin A (MitA), in A549 cells upregulated both SP1 and TS expression (Figure 5B). The SP1 inhibitor, mithramycin A was used to treat pemetrexed-resistant A400 cells and results showed that MitA dose-dependently (MitA), was used to treat pemetrexed-resistant A400 cells and results showed that MitA dose- suppressed the proliferation of A400 cells with or without pemetrexed treatment (Figure5C). dependently suppressed the proliferation of A400 cells with or without pemetrexed treatment (Figure 5C). MitA treatment in A400 or H1355 cells dose-dependently inhibited TS expression (Figure 5D).

Cancers 2020, 12, 2069 7 of 16

Cancers 2020, 12, x FOR PEER REVIEW 7 of 16 MitA treatment in A400 or H1355 cells dose-dependently inhibited TS expression (Figure5D). UsingUsing PTC-209 PTC-209 to to inhibit inhibit BMI1 BMI1 activation activation in inA400 A400 cells, cells, we wefound found that thatPTC-209 PTC-209 also alsoinhibited inhibited SP1 proteinSP1 protein expression expression (Figure (Figure 5E). We5E). further We further found found that there that therewas a was positive a positive correlation correlation between between BMI1 andBMI1 SP1 and mRNA SP1 mRNA expression expression in the in theCancer Cancer Genome Genome Atlas Atlas (TCGA) (TCGA) database database using using the the Genen Genen ExpressionExpression Profiling Proﬁling Interactive Interactive An Analysisalysis webtool webtool (GEPIA, (GEPIA, http://gepia.cancer-pku.cn/index.html) http://gepia.cancer-pku.cn/index.html (R) =( R0.64,= 0.64 p ,=p 0,= 0Figure, Figure 5F).5F). ItIt indicates indicates that that BMI1 BMI1 is is an an upstream upstream signaling signaling molecule molecule of Sp1 or TSTS expressionexpression in in pemetrexed pemetrexed NSCLC NSCLC cells. cells.

FigureFigure 5. 5. BMI1BMI1 is is an an upstream upstream signaling signaling molecule molecule of of SP SP11 protein protein expression expression in in pemetrexed-resistant pemetrexed-resistant NSCLCNSCLC cells. cells. (A (A) SP1) SP1 expression expression in inA549 A549 or orA400 A400 cell cellss was was determined determined by western by western blot. blot. (B) BMI1 (B) BMI1 and SP1and expression SP1 expression in tRFP- in tRFP- or BMI1-overexpressed or BMI1-overexpressed A549 A549(BS1) (BS1) were weredetermined determined by western by western blot. blot.(C) Mithramycin(C) Mithramycin A (MitA), A (MitA), the SP1 the SP1inhibitor, inhibitor, was wasused usedto treat to treatA400 A400 cells cellsat 50 at or 50 100 or nM 100 nMwith with co- treatmentco-treatment of pemetrexed of pemetrexed at the at indicated the indicated concentrat concentration.ion. DMSO DMSO was used was usedas a negative as a negative control. control. The cellThe survival cell survival was determined was determined by MTT by assay. MTT ( assay.D) A400 (D or) A400H1355 or cells H1355 were cells treated were with treated the indicated with the concentrationindicated concentration of MitA for of MitA48 h. forThe 48 protein h. The proteinexpression expression of SP1 ofor SP1 BMI1 or BMI1was then was thendetermined determined by westernby western blot. blot. (E) A400 (E) A400 cells cells were were treated treated with with the indicate the indicatedd concentration concentration of PTC-209 of PTC-209 for 48 for h 48and h andthe proteinthe protein expressions expressions of BMI1 of BMI1 or SP1 or SP1 were were determined determined by by western western blot. blot. (F ()F )The The correlation correlation between between mRNAmRNA of of SP1 SP1 and and BMI1 BMI1 among among lung lung adenocarcinoma adenocarcinoma patients patients of of the the Cancer Cancer Genome Genome Atlas Atlas (TCGA) (TCGA) databasedatabase was analyzedanalyzed by by the the Genen Genen Expression Expression Profiling Profiling Interactive Interactive Analysis Analysis webtool webtool (GEPIA) (GEPIA) website. website.The experiments The experiments of (A) to (ofE) ( wereA) to done(E) were three done times three and datatimes from and onedata experiment from one wereexperiment presented. were presented. 2.5. BMI1 Overexpression Activates EMT and Enhances Cancer Stemness in NSCLC Cells 2.5. BMI1It has Overexpression been suggested Acti thatvates cancer EMT chemoresistanceand Enhances Cancer could Stemness be induced in NSCLC by the Cells EMT program or be associatedIt has been with cancersuggested stemness. that cancer From chemoresistanc the results in Figuree could1C, anbe increasedinduced by EMT the processEMT program was found or be in associatedpemetrexed with A400 cancer cells instemness. comparison From to the parental results A549 in Figure cells. We1C, firstlyan increased examined EMT ifknockdown process was of found BMI1 ininfluences pemetrexed the A400 EMT processcells in comparison in A400 cells to and parental results A549 revealed cells. thatWe firstly the expression examined of if E-cadherin knockdown was of BMI1increased influences while the EMT transcriptional process in repressorsA400 cells ofand E-cadherin, results revealed such as that Snail1 the expression or ZEB1, were of E-cadherin decreased wasafter increased knockdown while of BMI1the transcriptional (Figure6A). We repressors next examined of E-cadherin, the changes such in theas Snail1 EMT processor ZEB1, in A549were decreasedcells after after overexpression knockdown of of BMI1. BMI1 By(Figure western 6A). blot We analysis, next examined the downregulation the changes in of the E-cadherin EMT process and inthe A549 upregulation cells after of overexpressi N-cadherinon was of simultaneouslyBMI1. By western observed blot analysis, in BMI1-overexpressed the downregulation A549 of cells E- cadherin(Figure6B), and which the upregulation suggests the activationof N-cadherin of the wa EMTs simultaneously program by BMI1. observed The upregulationin BMI1-overexpressed of Snail or A549ZEB1 cells was (Figure also observed 6B), which in BMI1-overexpressed suggests the activati A549on of cells the EMT (Figure program6B). In addition,by BMI1. theThe cell upregulation migration of Snail or ZEB1 was also observed in BMI1-overexpressed A549 cells (Figure 6B). In addition, the cell migration capability of A549 cells was also enhanced by BMI1 overexpression (Figure 6C). Using

Cancers 2020, 12, 2069 8 of 16

Cancers 2020, 12, x FOR PEER REVIEW 8 of 16 capability of A549 cells was also enhanced by BMI1 overexpression (Figure6C). Using tumorsphere tumorspherecultivation, the cultivation, overexpression the overexpression of BMI1 in A549 of BMI1 cells in increased A549 cells CSC increased activity CSC (Figure activity6D). Treatment (Figure 6D). of TreatmentPTC-209 in of pemetrexed-resistant PTC-209 in pemetrexed-resistant A400 cells inhibited A400 tumorspere cells inhibited number tumorspere in a dose number dependent in mannera dose dependent(Figure6E). Thesemanner data (Figure suggest 6E). that These BMI1 data overexpression suggest that in NSCLCBMI1 overexpression cells activates the in EMTNSCLC program cells activatesand cancer the stemness, EMT program which and may cancer lead tostemness pemetrexed, which resistance. may lead to pemetrexed resistance.

FigureFigure 6. 6. OverexpressionOverexpression of of BMI1 BMI1 induce inducess EMT EMT in in A549 A549 NSCLC NSCLC cells. cells. ( (AA)) A400 A400 cells cells were were transduced transduced withwith lentiviruses lentiviruses carrying carrying shLacZ, shLacZ, shBMI1#1, shBMI1#1, or or shBMI1#2, shBMI1#2, respective respectively,ly, for for 72 72 h h and and the the total total cellular cellular proteinsproteins were were collected collected for for western western blot blot analysis analysis of ofBMI1 BMI1 or orthe the EMT-related EMT-related molecules molecules including including E- cadherin/Snail1/ZEB1.E-cadherin/Snail1/ZEB1. GAPDH GAPDH was was used used as asa aprotein protein loading loading control. control. (B (B–D–D) )A549 A549 cells cells were were transducedtransduced withwith lentiviruseslentiviruses of of tRPF tRPF control control (A549-tRFP) (A549-tRFP) or flag-tagged-BMI1 or flag-tagged-BMI1 (A549-BS1) (A549-BS1) and selected and selectedwith 20 µwithg/mL 20 blasticidin µg/mL blasticidin S for 96 h.S for The 96 protein h. The expressions protein expressions of BMI1 andof BMI1 the EMT and markersthe EMT includingmarkers includingE-cadherin E-cadherin/N-cadherin/Snail1/ZEB/N-cadherin/Snail1/ZEB1 were determined1 were determined by western by blot western (B). Cell blot migration (B). Cell abilitiesmigration of abilitiesA549-tRFP of A549-tRFP or A549-BS1 or cells A549-BS1 were analyzed cells were by analyzed wound healing by wound assay healing (C). ** p assay< 0.01. (C The). **, CSC p < activity0.01. The of CSCA549-tRFP activity or of A549-BS1 A549-tRFP cells or were A549-BS1 assessed cells by were tumorsphere assessed cultivationby tumorsphere (D). The cultivation red fluorescent (D). The protein red fluorescent(RFP) signals protein (upper (RFP) panel) signals and tumorsphere (upper panel) numbers and (lowertumorsphere panel) were numbers captured (lower and countedpanel) were with capturedan inverted and fluorescence counted with microscopy an inverted at Day 14.fluorescence * p < 0.05. (microscopyE) Tumorsphere at Day cultivation 14. *, wasp < performed0.05. (E) Tumorsphereon A400 cells cultivation in the presence was performed of 1 or 2 µonM A400 PTC-209 cells andin the the presence formed of tumorspheres 1 or 2 µM PTC-209 were picturedand the formed(upper panel)tumorspheres and counted were (lowerpictured panel) (upper at Daypanel) 14. and ** p

2.6. PTC-209 Sensitizes NSCLC Tumors Toward Pemetrexed Treatment In Vivo

Cancers 2020, 12, 2069 9 of 16

2.6. PTC-209 Sensitizes NSCLC Tumors toward Pemetrexed Treatment In Vivo Cancers 2020, 12, x FOR PEER REVIEW 9 of 16 We finally examined the in vivo therapeutic potential of PTC-209 for the treatment of NSCLC tumorsWe with finally pemetrexed examined resistance. the in vivo The therapeutic pemetrexed-resistant potential of PTC-209 A400 NSCLC for the cells treatment were subcutaneously of NSCLC inoculatedtumors with into thepemetrexed back skin ofresistance. NOD/SCID The mice pemetrexed-resistant and divided into two A400 groups NSCLC when the cells tumor were volume reachedsubcutaneously 50 mm 3inoculated, with group into 1the as back the skin vehicle of NOD/SCID control and mice group and divided 2 as the into PTC-209-treated two groups when group, 3 withthe tumor 20 mg volume/kg for 5reached days/week 50 mm for, with a total group of 3 weeks.1 as the Aftervehicle one control week and of thegroup last 2 PTC-209as the PTC-209- treatment, alltreated the mice group, received with 20 100 mg/kg mg/kg for pemetrexed 5 days/week for for two a total times of with3 weeks. an interval After one of week 7 days of as the outlined last PTC- in our 209 treatment, all the mice received 100 mg/kg pemetrexed for two times with an interval of 7 days previous report [21]. The mice were then sacrificed at Day 7 after the last pemetrexed treatment and the as outlined in our previous report [21]. The mice were then sacrificed at Day 7 after the last tumor weight was measured. The weight of tumors was significantly lower in the PTC-209-pretreated pemetrexed treatment and the tumor weight was measured. The weight of tumors was significantly group (Figure7A). The percentage of tumor cells with BMI1 or Ki67, the marker for cell proliferation, lower in the PTC-209-pretreated group (Figure 7A). The percentage of tumor cells with BMI1 or Ki67, and the mean staining intensity of SP1 expression was significantly reduced in the PTC-209-pretreated the marker for cell proliferation, and the mean staining intensity of SP1 expression was significantly groupreduced (Figure in the7B). PTC-209-pretreated These results suggest group that (Figure PTC-209 7B). is These a potential results combination suggest that agent PTC-209 for NSCLCs is a treatmentpotential combination when pemetrexed agent for resistance NSCLCs has treatm developed.ent when pemetrexed resistance has developed.

FigureFigure 7. 7. PTC-209PTC-209 sensitizes sensitizes NSCLC tumors NSCLC toward tumors pemetrexed toward treatment pemetrexed in vivo. treatment Pemetrexed-in vivo. Pemetrexed-resistantresistant A400 cells were A400 subcutaneo cells wereusly subcutaneously inoculated into inoculated the back into skins the of back NOD/SCID skins of mice NOD as/SCID 2.5 × mice as10 2.56 cells/mouse106 cells (n/mouse = 4). After (n = tu4).mor After volume tumor reached volume to 50 reached mm3, the to mice 50 mm were3, divided the mice into were two divided groups: into × twogroup groups: 1: vehicle group (Cremophor/NaCl) 1: vehicle (Cremophor or group/NaCl) 2: PTC- or209 group treatment 2: PTC-209 with intrap treatmenteritoneal with injection intraperitoneal at a injectiondose of 20 at mg/kg a dose for of 5 20days/week, mg/kg for the 5treatments days/week, were the done treatments for a total were of 3 doneweeks. for After a total 7 days of from 3 weeks. Afterlast shot 7 days of vehiclefrom last or PTC-209, shot of vehicle all the or mice PTC-209, were treated all the micewith pemetrexed were treated at with a dose pemetrexed of 100 mg/kg at a dosefor of 100two mg times/kg forwith two an timesinterval with of an7 days. interval The ofmice 7 days. were The then mice sacrificed were thenat Day sacrificed 7 after the at Day last 7treatment after the last treatmentof pemetrexed. of pemetrexed. (A) Tumors (A )were Tumors harvested were harvested and the weight and the was weight compared was compared between betweenvehicle pre- vehicle pre-treatedtreated control control group group and and PTC-209 PTC-209 pre-treated pre-treated group. group. (B) Paraffin (B) Para ffiembeddedn embedded tumor tumor tissues tissues were were slicedsliced intointo 44 µµmm sections and and immunohistochemistry immunohistochemistry analysis analysis was was performed performed to evaluate to evaluate the protein the protein expressionexpression ofof BMI1,BMI1, SP1,SP1, andand Ki67.Ki67. The images we werere quantified quantified by by TissueFAX TissueFAX software software to to measure measure the positivethe positive percentages percentages ormean or mean staining staining intensity. intensity.

3.3. DiscussionDiscussion TheThe clinicalclinical useuse ofof pemetrexedpemetrexed waswas startedstarted inin treatingtreating mesothelioma in 2003 [30]. [30]. As As the the second- second-line treatmentline treatment for NSCLC, for NSCLC, pemetrexed pemetrexed offers offers clinically clinically equivalent equivalent effi cacyefficacy outcomes outcomes but but fewer fewer side side eff ects comparedeffects compared with docetaxel with docetaxel [31]. Based [31]. on Based better tolerabilityon better tolerability and more convenientand more administration,convenient platinumadministration, with pemetrexedplatinum with was pemetrexed suggested aswas standard suggested first-line as standard regimen first-line for advanced regimen NSCLC for in 2008advanced [32]. MaintenanceNSCLC in 2008 therapy [32]. Maintenance with pemetrexed therapy in with patients pemetrexed who received in patients the who preceding received induction the preceding induction therapy with platinum-pemetrexed revealed improved progression-free and overall survival in advanced NSCLC [33].

Cancers 2020, 12, 2069 10 of 16 therapy with platinum-pemetrexed revealed improved progression-free and overall survival in advanced NSCLC [33]. From the perspective of patients, not only the drug efficacy but also the quality of life is equally important for cancer patients. The properties of pemetrexed, such as good efficacy, convenient administration and fewer side effects, make it a widely acceptable drug among lung cancer patients. Reversing the pemetrexed resistance means patients can afford lower medical expenses and have a reasonable quality of life. Patients who developed drug resistance to pemetrexed may still have other treatment options, including second-line chemotherapy, tyrosine kinase inhibitors [34], or immunotherapy [35]. However, the above mentioned treatments are either too expensive or associated with lots of side effects. Using the combination of other drugs to make NSCLC patients regain their sensitivity to pemetrexed will have a positive impact on the financial burden of patients. The biochemical changes in tumor cells with chemoresistance include oncogene or oncoprotein regulations, enhanced DNA repair systems, activated EMT, and an increased number of CSCs [7,36]. In addition, the altered pharmacodynamics and pharmacogenetics of cancer cells also lead to chemoresistance. For example, the expression level of ATP-binding cassette transporters or multidrug resistance-associated proteins could facilitate the removal of anti-cancer drugs from cancer cells [37–39]. In addition, the polymorphisms of the enzymes involved in xenobiotic metabolisms, such as the members of the cytochrome P450 family [40], glutathione S-transferases, or diphosphate glucuronosyltransferases, have been frequently mentioned among the influences of pharmacokinetics of anti-cancer drugs [41,42]. Our study focuses on the oncogene BMI1 because it plays an important role in the development of tumor metastasis, stemness maintenance, and chemo- and radiotherapy resistance. Siddique et al. previously demonstrated that BMI1 was overexpressed in docetaxel-resistant prostate cancer cells [43]. Knockdown of BMI1 in docetaxel resistant prostate cancer cells inhibited the expression of cyclin D1 or BCL2, the downstream targets of the Wnt/β–catenin pathway. The overexpression of BMI1 in prostate cancer cells also enhanced the transcriptional activity of TCF [43]. The induction of the noncanonical Wnt pathway by BMI1 was also observed in melanoma cells that were resistant to the treatment of BRAF inhibitor by the upregulation of the Wnt5a and ROR2 receptors [44]. The overexpression of BMI1 in melanoma cells induced the invasive signature with concomitant nuclear accumulation of β-catenin [44]. Here, we described another pathway for BMI1-induced chemoresistance in NSCLCs through the upregulation of SP1 activity followed by the upregulation of TS, the elevated CSC activity, and turning on the EMT process. Using GEPIA to analyze the TCGA database, the mRNA expression between BMI1 and TYMS or SP1 and TYMS displayed a significantly positive correlation among lung adenocarcinoma patients (Figure S1, R = 0.42, p = 0 for BMI1/TYMS; R = 0.35, p = 3.6 10 15 for SP1/TYMS). Such observations from clinical data in the TCGA database × − strongly support our findings. SP1 is one of the transcription factors and belongs to the Sp/Krüppel like factor (KLF) family that plays multiple roles in maintaining cellular homeostasis and critical factors in diseases including cancer [45]. In lung cancer, SP1 expression is correlated with the tumor progression [46–48]. Wang HB, et al. found the core promoter regions of BMI1, and identified SP1 as an important transcription factor directly binding to the BMI1 promoter region in nasopharyngeal cancer [49]. However, our findings demonstrated that BMI1 could induce SP1 expression but inhibition of SP1 didn’t suppress the BMI1 activity in pemetrexed-resistant A400 cells (Figure S2). It indicates that SP1 is a downstream molecule of BMI1 activation in pemetrexed NSCLC cells. We found that the expression level of BMI1 was consistent with that of TS (Figure2B), which has been reported to be positively correlated to the pemetexed-resistance [50,51]. We observed that the treatment of PTC-209 in pemetrexed A400 cells enhanced the therapeutic effect of pemetrexed on A400 xenograft tumors in vivo (Figure7A). Yong et al. have demonstrated that the treatment of PTC-209 for one month in a mouse lung cancer model of C/EBPα (CCAAT/enhancer-binding protein α) deletion reduced the tumor burden by up to 70% [52]. PTC-596, an orally active BMI1 inhibitor, has been used for phase I evaluation in patients with advanced solid tumors (https://clinicaltrials.gov/ct2/show/NCT02404480). Cancers 2020, 12, 2069 11 of 16

We also observed the in vitro growth inhibition of Mithramycin A to pemetrexed A400 cells (Figure5C). Mithramycin A has been used for phase II evaluation in chest cancers including lung cancers (https://clinicaltrials.gov/ct2/show/NCT01624090). Our results indicate that pretreatment of PTC-209 could re-sensitize pemetrexed-resistant A400 tumors to pemetrexed (Figure7). It suggests the potentially clinical use of the inhibitors of BMI1 in NSCLC patients with pemetrexed resistance.

4. Materials and Methods

4.1. Chemicals and Reagents PTC-209, the BMI1 inhibitor, and mithramycin A, the SP1 inhibitor, were purchased from Cayman Chemicals (Ann Arbor, MI, USA) and dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) at 2 mM or 18 mM, respectively, and stored at 20 C. Pemetrexed disodium heptahydrate − ◦ was purchased from Eli Lilly and Company (Indianapolis, IN, USA) and dissolved in sterile ddH2O at 100 mg/mL and stored at 20 C. Puromycin and blasticidin S were purchased from TOKU-E − ◦ (Bellingham, WA, USA) and dissolved in PBS at 20 mg/mL and stored at 20 C. − ◦ 4.2. Cell Culture A549 or H1355 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and were maintained in Dulbecco’s Modiﬁed Eagle Medium (DMEM, GibcoTM, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, HyClone Laboratories Inc., South Logan, UT, USA) supplied with 1 mM glutamine (Gibco), 1 mM sodium pyruvate (Gibco), 1X Non-Essential Amino Acids (Gibco), and 100 U/mL penicillin/streptomycin (Gibco) at 37 ◦C, 5% CO2 incubator. A400 cells were derived from A549 cells with pemetrexed resistance, which were established as outlined in the previous report [21] and maintained in DMEM/10% FBS as used for A549 cells, except for the addition of 400 ng/mL pemetrexed.

4.3. Overexpression or Knockdown of BMI1 by Lentivirus Transduction The BMI1 gene was first amplified from A400 cDNA and cloned into p3XFlag-CMV-13 vector (Sigma-Aldrich) with the following primers: 50-CGCGGTACCATGCATCGAACAACGAGAA TC-30/50-AGCGGATCCACCAGAAGAAGTTGCTGATGAC-30 after KpnI and BamHI digestion. The flag-tagged BMI1 gene was further amplified and subcloned into pLAS5w.Pbsd-L-tRFP-C lentiviral vector (the National RNAi Core Facility at Academia Sinica, Taipei, Taiwan) with the following primers: 50-CGCGCTAGCATGCATCGAACAACGAGAATC-30/50-GCGTCTAGACTACTTGTCATCGTCATCC TTG-30 after NheI and XbaI digestion. BMI1-specific shRNA plasmids (TRCN0000020156 and TRCN0000229416) were obtained from the National RNAi Core Facility at Academia Sinica. The package of lentivirus was performed in 293T cells according to the protocol of our previous report [53]. The transduction of lentivirus was performed by the addition of 8 µg/mL polybrene (Sigma). For selection of successfully transduced tRFP/BMI1-tRFP or shRNAs, the cells were treated with 20 µg/mL blasticidin S or 2 µg/mL puromycin, respectively.

4.4. Western Blot Total cellular proteins were obtained by lyzed with RIPA buffer (Genetex Inc., Irvine, CA, USA) and the protein concentration was quantified with the Pierce™ BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). An amount of 25 µg of total cellular proteins was separated by SDS-PAGE and transferred onto polyvinylidene fluoride membrane (Pall Corporation, Washington, NY, USA). After blocking with 1% skim milk/TBS-T (20 mM Tris, 150 mM NaCl, 0.1% Tween-20) at room temperature (RT) for 1 h, the membrane was incubated with primary antibody at 4 ◦C overnight followed by horseradish peroxidase conjugated secondary antibody at RT for 1 h. The signal was then developed by Pierce™ ECL Western Blotting Substrate (Thermo Fisher) and captured by the FUSION Cancers 2020, 12, 2069 12 of 16

Solo S imaging system (Marne-la-Vallée, France). The antibodies used in this study were listed in Table S1.

4.5. Tumorsphere Cultivation Cells were harvested by trypsin/EDTA and were suspended at 2000 cells/2 mL/well of ultralow attachment 6-well-plate (Greiner Bio-One GmbH, Kremsmünster, Austria) at 37 ◦C/5% CO2 incubator in tumorsphere media at DMEM/F12 (Gibco) containing 0.4 mg/mL bovine serum albumin (Gibco), 0.01% methylcellulose (Sigma-Aldrich), 20 ng/mL epidermal growth factor (PeproTech, Inc., Rocky Hill, NJ, USA), 20 ng/mL basic ﬁbroblast growth factor (PeproTech), 5 µg/mL insulin (Sigma-Aldrich), 4 µg/mL heparin (Sigma-Aldrich), 1 µM hydrocortisone (Sigma-Aldrich). Wells were added with 300 µL of fresh tumorsphere media every 4 days and the formed primary tumorspheres were counted at Day 21 under inverted light microscopy (Motic Incorporation Ltd., Hong Kong) and collected with 100 µm cell strainer (BD Biosciences, Franklin Lakes, NJ, USA). After dissociation with HyQTase (Hyclone), 1000 cells from primary tumorspheres were used for secondary tumorsphere cultivation as the condition used for primary tumorspheres.

4.6. Wound Healing Assay Cell migration ability was determined by wound healing assay with a 2 well silicone insert (ibidi GmbH, Gräfelfing, Germany). Briefly, the silicone insert was placed in a 3.5 cm cell culture dish and loaded with 3 104 cells for attachment overnight. After removing silicone insert, the dish was × filled with complete culture medium and was pictured under an inverted light microscopy (Motic) before being put back into a 37 ◦C/CO2 incubator. The dish was pictured at 3, 6, 12, and 24 h and the cell migration area was analyzed by calculating the relative percentage of area without cell coverage using ImageJ software (Version 1.53a, National Institutes of Health, Bethesda, MD, USA).

4.7. NSCLC Xenograftment in NOD/SCID Mice The NSCLC xenograftment experiments in NOD/SCID mice were approved by the Institutional Animal Care and Use Committee in Chung Shan Medical University (Taichung, Taiwan) with the approval No. 2017. A400 cells (2.5 106) were suspended in 100 µL of 2.5 mg/mL matrigel × (BD Biosciences) and were subcutaneously injected at the right site of back part for tumor growth. As the tumor volume reached 50 mm3 by a calculation formula of 1/2 (length width2)[54], × the mice were divided into two groups; vehicle control (20% (w/w) Cremophor EL in 0.9% NaCl) and PTC-209/pemetrexed treatment. For the PTC-209/pemetrexed group, the tumor bearing mice firstly received PTC-209 at 20 mg/kg/day for 5 days a week for a total of 3 weeks and pemetrexed treatment was performed at 100 mg/kg once per week for a total of 2 weeks. The mice were sacrificed at Day 95 after injection of A400 cells and the xenografted tumors were harvested, weighted, and fixed with 3.7% formaldehyde (Sigma-Aldrich) for immunohistochemistry analysis.

4.8. Immunohistochemistrical Analysis The formaldehyde fixed tumor tissues were embedded into paraffin and sliced into 4 µm sections. After deparaffinization steps, the slides were incubated with primary antibodies at 4 ◦C overnight followed by HRP-conjugated secondary antibodies at room temperature for 1 h. After development with 3,30-diaminobenzidine substrate (VECTOR Laboratories, Inc., Burlingame, CA, USA), counter staining was performed on the slides with hematoxylin (Sigma-Aldrich) followed by scanning with the TissueFAXS PLUS system and quantification with TissueFAXS Imaging Software (version 7.0, TissueGnostics GmbH, Vienna, Austria). Cancers 2020, 12, 2069 13 of 16

4.9. Cell Cycle Analysis A400 (1.5 105) cells were seeded into 3.5 cm cell culture dishes and treated with PTC-209 or 0.1% × DMSO for 24 h followed by harvesting with trypsin/EDTA. After counting the cell number with trypan blue, 1 105 cells were used for fixation with 70% EtOH/PBS solution at 4 C overnight. The fixed × ◦ cells were then stained with 20 µg/mL PI in 0.1% Triton X-100/PBS at room temperature for 30 min. After being resuspended in 0.2 mg/mL RNase/PBS solution, the fluorescence signals of PI were collected Cancers 2020, 12, x FOR PEER REVIEW 13 of 16 by flow cytometry (BD FACSCalibur™, BD Biosciences, Franklin Lakes, NJ, USA) and the data were analyzed by FlowJo software (version 10, BD Biosciences). 4.10. Statistical Analysis 4.10. StatisticalQuantitative Analysis data were presented as the mean ± SD. The comparisons between the two groups wereQuantitative analyzed with data an were unpaired presented t-test. as The the meancomparisonsSD. The among comparisons multiple betweengroups (more the two than groups two) ± werewere analyzed analyzed with with an a unpairedrepeated tmeasure-test. The ANOVA comparisons followed among by multiplea Tukey–Kramer groups (morepost hoc than test two) to wereidentify analyzed differences with a among repeated specific measure groups. ANOVA A p followed value of by less a Tukey–Kramer than 0.05 was postconsidered hoc test statistically to identify disignificant.fferences among specific groups. A p value of less than 0.05 was considered statistically significant.

5.5. ConclusionsConclusions OurOur datadata revealed that that BMI1 BMI1 was was upregulat upregulateded in inpemetrexed-resistant pemetrexed-resistant NSCLC NSCLC cells. cells. The Theoverexpression overexpression of ofBMI1 BMI1 in inpemetrexed-sensitiv pemetrexed-sensitivee NSCLC NSCLC cells cells also also reduced reduced the eefficacyﬃcacy ofof pemetrexed.pemetrexed. The involvementinvolvement ofof BMI1BMI1 inin pemetrexedpemetrexed resistanceresistance maymay resultresult fromfrom threethree possiblepossible pathwayspathways including including (1) (1) the the upregulation upregulation of SP1 of followed SP1 followed by increasing by increasing TS expression; TS expression; (2) the induction (2) the ofinduction the EMT of process the EMT by process upregulating by upregulating Snail1 or ZEB1Snail1 expression; or ZEB1 expression; (3) the elevation (3) the elevation of CSC activity of CSC (Figureactivity8 ).(Figure It also 8). suggests It also suggests that the that inhibition the inhibition of BMI1 of BMI1 may may be a be potential a potential strategy strategy to to overcome overcome pemetrexedpemetrexed resistance resistance in in NSCLC NSCLC patients. patients.

Figure 8. The proposed model for BMI1 involved pemetrexted resistance in NSCLC. The role of BMI1Figure in 8. pemetrexed The proposed resistance model for could BMI1 result involved from pemetrex three waysted including resistance(1) in NSCLC. the upregulation The role of of BMI1 SP1 followedin pemetrexed by increased resistance TS expression; could result (2) from The three enhancement ways including of the EMT (1) the process upregulation by upregulating of SP1 followed Snail1 orby ZEB1 increased expression; TS expression; (3) The elevation(2) The enhancement of CSC activity. of the Inhibition EMT process of BMI1 by upregulating or SP1 activity Snail1 by PTC-209 or ZEB1 orexpression; mithramycin (3) AThe may elevation serve as of the CSC potential activity. agents Inhibition for overcoming of BMI1 or the SP1 pemetrexed activity by resistance PTC-209 inor NSCLCmithramycin patients. A may serve as the potential agents for overcoming the pemetrexed resistance in NSCLC patients.

SupplementarySupplementary Materials: Materials:The The following following are are available available online online at at http: www//www.mdpi.com.mdpi.com/xxx/s1./2072-6694 Figure /S1:12/ 8Inhibition/2069/s1. Figure S1: Inhibition of SP1 activity did not inﬂuence BMI1 expression in pemetrexed-resistant NSCLC cells; of SP1 activity did not influence BMI1 expression in pemetrexed-resistant NSCLC cells; Figure S2: The positive Figure S2: The positive correlation between BMI1/TYMS or SP1/TYMS was found in the TCGA database; Table S1: Antibodiescorrelation used between in this BMI1/TYMS study. or SP1/TYMS was found in the TCGA database; Table S1: Antibodies used in this study.

Funding: This research was funded by the Ministry of Sciences and Technology in Taiwan (grant No. MOST 107-2314-B-371-012 by B.-Y.W.; grant No. MOST 106-2314-B-040-024-MY3 by W.-W.C.).

Acknowledgments: We thank the National RNAi Core Facility at Academia Sinica in Taiwan for providing shRNA reagents and related services.

Cancers 2020, 12, 2069 14 of 16

Author Contributions: Conceptualization; H.-T.S., B.-Y.W. and W.-W.C.; Data curation: P.-J.C. and S.-H.C.; Data analysis: G.-T.S., P.-J.C., S.-H.C., and W.-W.C.; Resources: M.-S.J., G.-T.S., and W.-W.C.; Writing—original draft: H.-T.S., M.-S.J. and W.-W.C.; Writing—review & editing: B.-Y.W. and W.-W.C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Ministry of Sciences and Technology in Taiwan (grant No. MOST 107-2314-B-371-012 by B.-Y.W.; grant No. MOST 106-2314-B-040-024-MY3 by W.-W.C.). Acknowledgments: We thank the National RNAi Core Facility at Academia Sinica in Taiwan for providing shRNA reagents and related services. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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