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Oxidative stress enhances tumorigenicity and stem-like features via the activation of the Wnt/β-catenin/MYC/Sox2… Wu, Chengsheng; Gupta, Nidhi; Huang, Yung-Hsing; Zhang, Hai-Feng; Alshareef, Abdulraheem; Chow, Alexandra; Lai, Raymond Apr 2, 2018

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RESEARCH ARTICLE Open AccessOxidative stress enhances tumorigenicityand stem-like features via the activation ofthe Wnt/β-catenin/MYC/Sox2 axis in ALK-positive anaplastic large-cell lymphomaChengsheng Wu1,6, Nidhi Gupta1†, Yung-Hsing Huang1†, Hai-Feng Zhang1,4, Abdulraheem Alshareef1,5,Alexandra Chow1 and Raymond Lai1,2,3*AbstractBackground: The phenomenon that malignant cells can acquire stemness under specific stimuli, encompassed underthe concept of cancer cell plasticity, has been well-described in epithelial malignancies. To our knowledge, cancer cellplasticity has not yet been described in hematopoietic cancers. To illustrate and study cancer cell plasticity inhematopoietic cancers, we employed an in-vitro experimental model of ALK-positive anaplastic large-cell lymphoma(ALK+ALCL) that is based on the phenotypic and functional dichotomy of these cells, with cells responsive to a Sox2reporter (i.e. RR cells) being significantly more stem-like than those unresponsive to the reporter (i.e. RU cells).Methods: H2O2 was employed to trigger oxidative stress. GFP expression and luciferase activity, readouts of the Sox2reporter activity, were quantified by using flow cytometry and luciferase activity assay, respectively. Doxorubicin-resistance and clonogenicity were assessed by using the MTS, methylcellulose colony formation and limiting dilutionassays. Western blotting and quantitative PCR were used to assess the expression of various members of the Wnt/β-catenin pathway. Pull-down studies using a Sox2 binding consensus sequence were used to assess Sox2-DNA binding.Quercetin and 10074-G5 were used to inhibit β-catenin and MYC, respectively. siRNA was used to downregulate Sox2.Results: Under H2O2-induced oxidative stress, a substantial fraction of RU cells was found to convert to RR cells, asevidenced by their acquisition of GFP expression and luciferase activity. Compared to the native RU cells, converted RRcells had significantly higher levels of doxorubicin-resistance, clonogenicity and sphere formation. Converted RR cellswere characterized by an upregulation of the Wnt/β-catenin/MYC/Sox2 signaling axis, previously found to be the keyregulator of the RU/RR dichotomy in ALK+ALCL. Furthermore, Sox2 was found to bind to DNA efficiently in convertedRR cells but not RU cells, and this finding correlated with significant elevations of several Sox2 downstream targetssuch as WNT2B and BCL9. Lastly, inhibition of β-catenin, MYC or Sox2 in RU cells significantly abrogated the H2O2-induced RU/RR conversion.Conclusions: We have demonstrated that cancer cell plasticity exists in ALK+ALCL, a type of hematopoietic cancer. Inthis cancer type, the Wnt/β-catenin/MYC/Sox2 axis is an important regulator of cancer cell plasticity.Keywords: Anaplastic large-cell lymphoma, Oxidative stress, Cancer cell plasticity, Intra-tumoral heterogeneity* Correspondence: rlai@ualberta.ca†Equal contributors1Department of Laboratory Medicine and Pathology, University of Alberta,5142J Katz Group Centre for Pharmacy and Health Research, Edmonton, ABT6G 1Z2, Canada2Department of Oncology, University of Alberta, Edmonton, AB, CanadaFull list of author information is available at the end of the article© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Wu et al. BMC Cancer  (2018) 18:361 https://doi.org/10.1186/s12885-018-4300-2BackgroundThe biological and clinical significance of intra-tumoralheterogeneity have been increasingly recognized [1].Some degree of intra-tumoral heterogenicity is believedto be resulted from clonal evolution, a process in whichindividual cancer cell clones gradually acquire an in-creasing number of genetic defects and the subsequentselection of cell clones carrying highly aggressive ormetastatic phenotype [2]. Intra-tumoral heterogeneity isalso likely linked to the concept of cancer stem cells(CSCs), which represent a very small population withinthe tumors that are capable of self-renewal as well asgeneration of various elements of the tumors [1, 2]. Inrecent years, accumulating evidence has suggested thatsome of the bulk tumor cells can acquire cancer stem-ness under certain circumstances or specific stimulations[1–3]. This phenomenon is encompassed under the con-cept of “acquired stemness’ or cancer cell plasticity [4].Thus far, cancer cell plasticity has been largely describedand studied in epithelial and neurogenic malignancies.For instance, MCF7, an estrogen receptor-positive breastcancer cell line, was found to acquire higher levels oftumorigenic potential and chemoresistance upon oxidativestress [5]. In another study, it was found that hypoxicstress can result in the enrichment of CSCs in gliomas,which can be identified by means of their high CD133 ex-pression [6]. To our knowledge, cancer cell plasticity hasnot been described in hematologic malignancies.ALK-positive anaplastic large-cell lymphoma (ALK+ALCL) is a distinct form of T-cell non-Hodgkin lymph-oma recognized in the World Health Organization Clas-sification of Hematopoietic Neoplasms [7]. The majorityof these tumors are found to carry the characteristic re-ciprocal chromosomal translocation involving ALK andNPM, and the resulted fusion gene product, NPM-ALK[7]. NPM-ALK has been shown to be the key oncogenicdriver of ALK+ALCL [8]. A large body of experimentaldata has suggested that NPM-ALK mediates its onco-genic potential by exerting its constitutively active tyro-sine kinase activity on a host of cellular signalingproteins, including those in the JAK/STAT, MAPK/ERK,and PI3K/AKT pathways, resulting in their inappropriateactivation [8]. In our prior studies of ALK+ALCL, wehad identified two distinct cell subsets in ALK+ALCLbased on their differential response to a commerciallyavailable Sox2 reporter, which key components consistof the Sox2 regulatory region 2 (SRR2) as well as twogene reporters (Green Fluorescence Protein, GFP andFirefly luciferase) [9]. Specifically, we found that reporterresponsive (RR) cells are significantly more tumorigenicand stem-like than reporter unresponsive (RU) cells. In arecently published manuscript, we also have reportedthat the Wnt/β-catenin/MYC/Sox2 signaling axis is thekey regulator of the RU/RR dichotomy in ALK+ALCL,with this axis being substantially more active in RR cellsthan RU cells [10]. Due to the inclusion of the GFP genein the Sox2 reporter, RU and RR cells present in ALK+ALCL cell lines can be easily detected and purified byusing flow cytometry.In this study, we found that a fraction of RU cells canconvert to RR cells upon oxidative stress, and this con-version was associated with the acquisition of cancerstem-like features. We believe that these observationsexemplify cancer cell plasticity in a hematologic malig-nancy. Our study results also have provided further sup-port that the Wnt/β-catenin/MYC/Sox2 axis is the keyregulator of cancer stemness in ALK+ALCL.MethodsCell lines and chemicalsKarpas 299, an ALK+ALCL cell lines, was a kind giftfrom Dr. Marshall Kadin (Boston, MA). SupM2, anotherALK+ALCL cell lines, was purchased from DeutscheSammlung von Mikrooorganismen und ZellkulturenGmbH (DSMZ), Germany (catalog number, ACC 509).Karpas 299 and SupM2 cell lines were grown in RPMI1640 medium (Invitrogen, Life Technologies, Grand Is-land, NY) with supplementations of 10% fetal bovineserum (Invitrogen), 1% Penicillin/Streptomycin (Ther-moFisher Scientific, Burlington, Ontario, Canada) and2 μg/mL puromycin dihydrochloride (Sigma Aldrich, StLouis, MO) in 5% CO2 atmosphere at 37 °C. Chemicalsused in this study, including hydrogen peroxide (H2O2),doxorubicin, N-acetyl-L-cysteine (NAC), iodonitrotetra-zolium chloride, 10074-G5 and quercetin, were pur-chased from Sigma Aldrich.Flow cytometric isolation of RU and RR cellsRU and RR cells derived from SupM2 and Karpas 299were purified with the use of a flow cytometry cell sorter(Becton Dickinson Biosciences, Franklin Lakes, NJ), anddetails of the method have been described previously [10].Luciferase reporter assay and flow cytometryThe luciferase reporter activity was measured by usingthe luciferase assay kit (Promega, Madison, WI), and theprocedure was in accordance with the manufacturer’sprotocol. The performance of flow cytometry has beendetailed in a previous publication [9]. A software (FCSExpress 5) from De Novo Software (Glendale, CA) wasused to analyze the generated flow cytometry data.Trypan blue exclusion and MTSTrypan blue exclusion and the MTS were used to quan-tify the number of viable cells (i.e. cell growth), and de-tails of these assays have been previously described [11].Wu et al. BMC Cancer  (2018) 18:361 Page 2 of 11Oxidative stress treatmentSupM2 or Karpas 299 cells growing in the log phasewere seeded into 25 mL Corning™ cell culture flask(ThermoFisher Scientific Canada). For the H2O2 re-challenge experiments, cells were initially treated withtwo different doses of H2O2 (0.3 or 0.5 mM) for 3 days.On day 3, cells were subjected to centrifugation (50 X g,10 min, room temperature), washed and cultured infresh culture medium supplemented with either 0.3 mMor 0.5 mM H2O2 for two additional days (i.e. day 4 andday 5). At the end of the experiments, we performed lu-ciferase reporter assay and flow cytometry to evaluatethe luciferase activity and GFP expression, respectively.For the N-acetyl-L-cysteine (NAC) experiments, RUcells derived from SupM2 were treated with 0.3 mMH2O2 in the presence of 0–20 mM of NAC for 2 days,and these cells were assessed for their SRR2 reporter ac-tivity (i.e. luciferase activity and GFP expression). Forthe 10074-G5 or quercetin experiments, ALK+ALCLcells were treated with 5 μM of 10074-G5 or 50 μM ofquercetin from day 4 to day 5 (24 h in total).Short interfering RNA and transfectionShort interfering RNA (siRNA, SMART pool, Dharma-con, Lafayette, CO) against Sox2 was used to down-regulate Sox2. Cells transfected with scrambled siRNA(Dharmacon) were included as the negative controls.BTX Electro Square ECM830 (225 V, 8.5 milliseconds, 3pulses) was used to transfect siRNA species into ALK+ALCL cells. The efficiency of siRNA knockdown ofSox2 was assessed by using Western blotting.RNA extraction, cDNA synthesis, and quantitative reversetranscriptase PCR (qRT-PCR)The method has been described previously [10]. Primersequences used in this study were listed in Table 1.Western blottingDetails of the method have been described previously [11].Antibodies reactive to phosphorylated ALKY1604 (catalog#3341), ALK (D5F3®), phosphorylated STAT3Y705 (D3A7),STAT3 (124H6), phosphorylated MYCS62 (E1J4K), MYC(D84C12), Sox2 (D6D9), and β-catenin (D10A8) were pur-chased from Cell Signaling Technology (Danvers, MA).Antibody reactive to β-actin (sc-130300) was purchasedfrom Santa Cruz (Dallas, TX), and antibody against activeβ-catenin (8E7) was from Merck Millipore (Toronto, ON,Canada). Anti-mouse IgG (catalog #7076) and anti-rabbitIgG (catalog #7074) were from Cell Signaling. Densitom-etry data was analyzed using Imag J software (National In-stitutes of Health, Bethesda, MD), and the values werenormalized to those of β-actin or γ-tubulin.SRR2 probe binding assayDetails of this method have been described previously[10]. The sequence of the SRR2 probe is as follows:5‘-AAGAATTTCCCGGGCTCGGGCAGCCATTGT-GATGCATATAGGATTATTCACGTGGTAATG-3′.The underlined sequence is the SRR2 consensussequence.Methylcellulose colony formation assayThe methylcellulose-based media were purchased fromthe R&D Systems Inc., (Minneapolis, MN). For each ex-perimental group, we seeded 500 cells in each well, asdescribed previously [11]. After ~ 10 days of culture, weemployed iodonitrotetrazolium chloride to stain the col-onies. Only colonies with > 40 cells were counted. Tripli-cate experiments were done. Images of the colonieswere acquired using the Alphalmager HP (ThermoFisherScientific Canada).Limiting dilution assayALK+ALCL cells, with or without H2O2 treatment, wereseeded in RPMI1640 (Invitrogen) supplemented with20% fetal bovine serum (Invitrogen), 1% penicillin/streptomycin and 2 μg/mL puromycin dihydrochloride(Sigma Aldrich) in 96-well low adherent plate (Corning).Ten different cell numbers, ranging from 1 to 1000 cellswere used. After 7 days of culture, cells were stainedwith iodonitrotetrazolium chloride (Sigma Aldrich) for24 to 48 h and images were acquired by using Alphalma-ger HP (ThermoFisher Scientific Canada). Cell spheresvisible to naked eyes were counted. Triplicate experi-ments were used.Statistical analysisThe generated data in all quantitative assays of this studywas presented as mean ± standard deviation. GraphPadPrism 5 software (La Jolla, CA) was employed forTable 1 The primer sets for qRT-PCR performed in this studyGene Forward Primers Reverse PrimersBCL9 5’-GGCCATACCCCTAAAGCACTC-3’ 5’-CGGAAATACTTCGCTCCTTTT-3’CTNNB1 5′- AAAGCGGCTGTTAGTCACTGG-3’ 5′- CGAGTCATTGCATACTGTCCAT-3’SOX2 5′- GCCGAGTGGAAACTTTTGTCG-3’ 5′- GGCAGCGTGTACTTATCCTTCT-3’WNT2B 5′- GATCAAGATGGTGCCAACTTC-3’ 5′- CCAAGACACAGTAATCTGGAGAG-3’GAPDH 5′- GGAGCGAGATCCCTCCAAAAT-3’ 5′- GGCTGTTGTCATACTTCTCATGG-3’Wu et al. BMC Cancer  (2018) 18:361 Page 3 of 11statistical analysis. The statistical significance of the dif-ferences between two experimental groups of test sam-ples was assessed by using Student’s t-test. Statisticalsignificance is denoted by * (p < 0.05) or ** (p < 0.01).ResultsOxidative stress induces a conversion from RU to RR cellsIn two of our recently published studies, we describedthat subsets of RU cells derived from esophageal squa-mous cell carcinoma and breast cancer cells can convertto RR cells when they were exposed to relatively lowconcentrations of H2O2, an agent known to generate re-active oxygen species (ROS) and induce potent oxidativestress [12, 13]. In this study, we asked if the H2O2-in-duced RU/RR conversion also exists in hematopoieticcancers such as ALK+ALCL. Two well-established ALK+ALCL cell lines, SupM2 and Karpas 299, were used. Asshown in Fig. 1a, 2 days after H2O2 treatment (0–1.0 mM) of purified RU cells, GFP-positive cells becamedetectable by flow cytometry, with ~ 55% ‘converted RR’cells at 0.5 mM of H2O2 for SupM2 and ~ 20% ‘con-verted RR’ cells at 1 mM of H2O2 for Karpas 299.Since high doses of H2O2 are cytotoxic, we attemptedto determine an optimal dose of H2O2 at which we cangenerate the highest number of viable converted RR cellsfor our studies. As shown in Additional file 1: Figure S1,we found this optimal H2O2 level to be 0.3 mM forSupM2, with which we generated ~ 30 viable convertedRR on day 2, for ~ 70 RU cells used at the beginning ofthe experiment. For Karpas 299, this optimal dose wasdetermined to be 0.5 mM H2O2, with which we gener-ated ~ 40 viable converted RR cells on day 2, for ~ 100RU cells used at the beginning of the experiment. Tofurther increase the yield, we re-challenged ALK+ALCLcells with H2O2 on day 3. As shown in Fig. 1b-c, this ex-perimental manipulation resulted in ~ 60% GFP-positive,converted RR cells derived from SupM2 and ~ 35% viableGFP-positive, converted RR cells derived from Karpas 299.This H2O2 stimulation protocol was used consistently forthe remainder of this study. Correlating with these flowcytometry results, the luciferase activity in converted RRcells was significantly higher than that of native RU cellsderived from both cell lines (Fig. 1d). Of note, native RRcells derived from SupM2 also showed significantly higherreporter activity (i.e. luciferase and GFP expression) uponH2O2 stimulation (Fig. 1d-e).To confirm that the RU to RR conversion induced byH2O2 was directly linked to the cellular response inducedby oxidative stress, we examined if the addition of N-acetyl-L-cysteine (NAC), a pharmacologic agent known tominimize cellular oxidative stress [14], can abrogate theconversion. As shown in Additional file 2: Figure S2A-B,in a dose-dependent manner, NAC significantly loweredthe number of H2O2-induced GFP-positive cells as well asluciferase activity.Converted RR cells are phenotypically similar to native RRcellsTo address the question of whether converted RR cellsare phenotypically similar to native RR cells, we com-pared these two types of cells with respect to chemo-resistance, clonogenicity and sphere-forming ability.First, to evaluate chemo-resistance, we subjected nativeRU and converted RR cells derived from SupM2 to vari-ous doses of doxorubicin, a chemotherapy drug com-monly used to treat ALK+ALCL patients [7]. As shownin Fig. 2a, we found that converted RR cells exhibitedsignificantly higher resistance to doxorubicin comparedto native RU cells. Expectedly, RR cells stimulated withH2O2 were also significantly more resistant to doxorubi-cin than native RR cells at 200 ng/mL of doxorubicin.Next we compared native RU and converted RR cellswith respects to their clonogenicity by employing themethylcellulose colony formation assay. As shown inFig. 2b, we found that converted RR cells derived fromSupM2 formed significantly more colonies than nativeRU cells (113 ± 7 versus 74 ± 9, p = 0.008). This differ-ence was observed even in the presence of doxorubicin.In comparison, when comparing native RR cells and RRcells stimulated with H2O2, we found that oxidativestress resulted in a significant increase in colony forma-tion, but only in the presence of doxorubicin (Fig. 2b).We then performed a comparison between native RUcells and converted RR cells regarding their sphere forma-tion ability in a limiting dilution manner. As illustrated inFig. 2c, we seeded increasing numbers of cells (i.e. 1 to1000) in each of the wells in 96-well plates, and the lowestcell numbers in which colonies were visible by naked eyeswere determined for native RU and converted RR cells.For SupM2, we found that a statistically significant differ-ence between native RU and converted RR cells was foundin 8 cells seeded in the wells. For Karpas 299, we foundthat a significant difference between native RU andconverted RR cells was at 125 cells seeded in the wells(Additional file 3: Figure S3). We also compared native RRcells and RR cells stimulated with H2O2; a significant dif-ference was found at 32 cells seeded in the wells for Kar-pas 299 cells (Fig. 2c and Additional file 3: Figure S3). Thesame experiment was performed using SupM2; no signifi-cant difference was observed.Lastly, we assessed the growth rates of native RU cells,converted RR cells, native RR cells and RR cells stimulatedwith H2O2. As shown in Additional file 4: Figure S4, nosignificant differences were found. This result strongly ar-gues against that the observed phenotypic differences be-tween native RU and converted RR cells, as well asbetween native RR cells and H2O2-stimulated RR cellsWu et al. BMC Cancer  (2018) 18:361 Page 4 of 11were simply due to a substantial difference in their growthrates.The Wnt/β-catenin/MYC/Sox2 axis is active in bothconverted RR cells and native RR cellsWe recently published that the Wnt/β-catenin/MYC/Sox2axis is a key regulator of the RU/RR dichotomy [10]. Specif-ically, inhibition of this axis abrogated the RR phenotypeand significantly decreased their clonogenicity and stem-like features [10]. In this study, we questioned if the H2O2-induced RU/RR conversion correlates with an activation ofthis signaling axis. As shown in Fig. 3a, compared to nativeRU cells, converted RR cells derived from SupM2 cellsshowed higher levels of active β-catenin, total β-cateninand p-MYCS62 (the active form of MYC) [15]. The sameexperiments were repeated using Karpas 299 cells and wefound similar findings. In comparison, we did not identifyany appreciable differences in the expression and activationof NPM-ALK or STAT3 between native RU and convertedRR cells (Additional file 5: Figure S5).To substantiate our finding that the Sox2 transcrip-tional activity was upregulated during the H2O2-inducedRU/RR conversion, we employed quantitative real time-PCR (qRT-PCR) to assess the expression of several Sox2abdcdFig. 1 Oxidative stress induces the conversion of RU to RR cells. a RU cells derived from SupM2 and Karpas 299 cells were subjected to various dosesof H2O2 for up to 2 days. The percent of GFP-positive cells was assessed by flow cytometry in RU cells from day 0 (the day of the treatment), day 1,and day 2. b The schematic experimental model of 0.3 mM H2O2 re-challenge treatment in RU cells derived from SupM2. c The GFP-positive cells inRU cells derived from SupM2 and Karpas 299 cells upon 0.3 mM and 0.5 mM H2O2 re-challenge treatment, respectively, at day 5. d The SRR2 luciferaseactivity in both RU and RR cells derived from SupM2 and Karpas 299 cells upon H2O2 re-challenge treatment at day 5. e The GFP-intensity of RU cellsderived from SupM2, RU cells with H2O2 re-challenge, RR cells and RR cells with H2O2 re-challenge at day 5Wu et al. BMC Cancer  (2018) 18:361 Page 5 of 11downstream targets. As shown in Fig. 3b, the mRNA ex-pression levels of WNT2B and BCL9 were significantlyhigher in converted RR cells as compared to native RUcells (Fig. 3b). Similar results were observed when wecompared native RR cells with RR cells stimulated withH2O2 (lower panel in Fig. 3b). Furthermore, we performedthe Sox2-SRR2 probe binding assay. As shown Fig. 3c,substantially more Sox2 protein was pulled down by thebiotin-labeled SRR2 probe in converted RR cells as com-pared to native RU cells. Furthermore, an appreciable in-crease of Sox2-SRR2 binding was identified in RR cellstreated with H2O2 (Fig. 3c). Notably, the Sox2 proteinlevel in both RU and RR cells derived from SupM2 wasnot appreciably changed in these experiments (Fig. 3c).Blockage of Wnt/β-catenin/MYC/Sox2 axis abrogates theRU to RR conversionNext, we determined if the inhibition of the Wnt/β-ca-tenin/MYC/Sox2 axis can significantly abrogate the RU/RR conversion induced by H2O2. To achieve this goal,we treated RU cells with quercetin (a β-catenin inhibi-tor) or 10074-G5 (a MYC inhibitor that can abrogateMYC-MAX heterodimerization and their DNA binding)[16] during the process of H2O2 challenge, as describedin Methods and Materials. As shown in Fig. 4a, pharma-cological inhibition of MYC or β-catenin dramaticallydecreased the expression level of MYC detectable byWestern blots, and significantly reduced the luciferaseactivity in RU cells stimulated with H2O2. When we per-formed siRNA to knockdown Sox2 in RU cells fromboth ALK+ALCL cell lines, we found a significant de-crease in the RU/RR conversion, as evidenced by thefinding that the H2O2-induced increases in the luciferaseactivity were significantly attenuated (Fig. 4b). Inaddition, we found that siRNA knockdown of Sox2 innative RU cells derived from SupM2 also largelyabrogated the H2O2-induced chemo-resistance to doxo-rubicin (Additional file 6: Figure S6).a bcFig. 2 Converted RR cells share similar biological functions with native RR cells. a RU and RR cells from SupM2 cells were re-challenged with 0.3 mMH2O2 for 5 days. Then the cells were subjected to 0, 100, 200 ng/mL doxorubicin for 48 h. Cells without H2O2 treatment were included as control. b RUand RR cells derived from SupM2 cells were re-challenged with 0.3 mM H2O2 for 5 days. Then the cells were processed for methylcellulose colonyformation assay in the presence/absence of 50 ng/mL doxorubicin, cells without H2O2 treatment were included as control. The graph demonstratedthe number of colonies in the above experimental groups. The lower panel showed one representative result of triplicate experiments. Only colonywith 40 cells (as pointed by the black arrow) or more was counted. c The serial diluted RU and RR cells derived from SupM2 wereseeded in 96-well plate. After ~ 8 days, the number of spheres was counted in the highlighted wells circulated by the rectangle lines.The right panel showed the analyzed results which indicated that converted RR cells and native RR cells have formed more spheres in alower number of cells seeded, in comparison with native RU cellsWu et al. BMC Cancer  (2018) 18:361 Page 6 of 11DiscussionIn several recent reviews, the concepts and significanceof cancer cell plasticity and acquired cancer stemnesshave been eloquently explained [2, 17, 18]. One of thekey elements of these concepts is that the CSC is a dy-namic phenotype, which can be acquired by non-stemcells if appropriate stimuli and microenvironment areprovided. Virtually all of the published studies supportingthese concepts are based on experimental data derivedfrom malignant epithelial and neural-derived cells, andthe induction of cancer stem-like features often involvesthe use of various chemotherapeutic agents, radiationtherapy, hypoxia and oxidative stress [2, 17, 19, 20]. Forinstance, the size of the CSC population in head and necksquamous carcinoma cells, detectable by their ALDHhigh/CD44high phenotype, was found to be significantlyincreased when these cells were exposed to cisplatin [21].In two other studies, human pancreatic cancer cellssubjected to a fibrogenic microenvironment were found toacquire CSC-like features [22, 23]. Hypoxia-induciblefactors (HIFs), which exists in specific tumor microenvir-onment niche, is also known to promote metastasis,tumorigenicity and CSC-like features in certain humancancer models [24, 25].a bcFig. 3 Converted RR cells share similar biochemical features with RR cells. a The Western blotting results showed the protein levels of activeβ-catenin, total β-catenin, p-MYCS62, and MYC in RU and RR cells derived from SupM2 and Karpas 299 cells, with or without H2O2 re-challenge. bThe qRT-PCR results suggested that the Sox2 downstream targets including WNT2B and BCL9 significantly increased in mRNA level in convertedRR cells (upper panel) and RR cells re-challenged with H2O2 (lower panel), in comparison with their counterpart cells. c SRR2 probe pull-downassay was performed to evaluate the Sox2-SRR2 DNA binding ability. The results suggested that more Sox2 was pulled down by biotin-labeledSRR2 probe in converted RR cells and RR cells after H2O2 stimulation as compared to their native RU and RR cells. The input of the pull-downassay demonstrated that the Sox2 expression was not appreciably altered in this experiment. Imag J software was utilized to analyze thedensitometry. Densitometry values of proteins of interest were all normalized to β-actin bands, and the densitometry value of protein ofinterest in RU cells was normalized as 1.0Wu et al. BMC Cancer  (2018) 18:361 Page 7 of 11The biochemical basis of how cancer cells acquirestemness is not completely understood, although there isevidence that activation of specific signaling pathways ortranscription factors is likely important. In this regard,Saijo et al. have reported that oxidative stress-inducedde-differentiation of lung cancer cells into CSCs is medi-ated via the activation of HOXA5 and upregulation ofSox2 [26]. In another study, hypoxia, which is also an in-ducer of oxidative stress [27], was found to increase theproportion of CD133-positive CSCs in glioma throughthe activation of the PI3K/AKT and ERK pathways [6]. Amore recent study has shown that the tumor-propagating potential of glioblastoma cells can be en-hanced by increasing the expression of a set of transcrip-tion factors normally involved in neuronal development,including POU3F2, SOX2, SALL2 and OLIG2 [28].Epithelial-to-Mesenchymal Transition (EMT), which canbe linked to the acquisition of cancer stemness [29, 30],is also known to be regulated by a number of cellularsignaling pathways. For instance, activation of the RAS-MAPK pathway was found to induce EMT and increasethe size of the CSC-like cell population [31]. Chaffer etal. have shown that non-CSCs derived from humanbreast cancers (CD44low) can convert to CSC’s(CD44high), and this conversion is mediated by ZEB1, animportant EMT transcription factor [32]. The samegroup also demonstrated that TGFβ collaborates withthe canonical and non-canonical Wnt signaling path-ways to induce EMT [33].Studies of acquired cancer stemness require appro-priate experimental models. Most of the publishedstudies in this field measured how various experimen-tal manipulations change the size of the CSC popula-tions, which are typically detected based on theirexpression of specific cell surface markers such asCD133 and CD44 [12, 20, 34–38]. In our studies, weemployed a different approach. Specifically, the CSC-like population was defined, detected and isolatedbased on their responsiveness to a Sox2 reporter. Inthe literature, we are aware of a number of recenta bFig. 4 Blockage of Wnt/β-catenin/MYC/Sox2 axis abrogates the RU to RR conversion. a Pharmacological inhibition of MYC or β-catenin using5 μM 10074-G5 or 50 μM quercetin for 24 h significantly decreased the SRR2 luciferase activities that were upregulated by H2O2 re-challenge. TheWestern blots below showed the knockdown efficiency of MYC and β-catenin after inhibitor treatment for 24 h. b RU cells with Sox2 knockdownby siRNA showed significantly decreased SRR2 luciferase activity in comparison to cells transfected with scrambled siRNA in the H2O2 re-challengeexperiment. The Western blots below suggested the Sox2 knockdown efficiency at 48 h post siRNA transfection. Imag J software was used toanalyze the densitometry. All proteins’ densitometry values were normalized to γ-tubulin bands, and the densitometry value of protein of interestin RU cells was normalized as 1.0Wu et al. BMC Cancer  (2018) 18:361 Page 8 of 11publications in which the CSC populations are alsoidentified based on their differential responsiveness tothe same Sox2 reporter used in this current study[13, 39–42]. In addition to the Sox2 reporter, we alsofound one recent publication in which the CSC popu-lation from colorectal cancer was identified based ontheir differential response to a Wnt reporter [38].Similar to our RU/RR model, Wnt reporter responsivecells were found to be more stem-like than unresponsivecells, and a very small subset of reporter unresponsivecells were found to convert into reporter responsive cellsupon stimulation with hepatocyte growth factor [38].Taken together, we believe that the use of differential re-porter responsiveness is a valuable experimental approachto study the acquisition of cancer stemness. In contrastwith the use of cell surface markers, we believe that thisexperimental approach has certain advantages, as it pro-vides direct clues to the biochemical mechanisms regulat-ing the cancer stemness phenotype.From our literature search, the concept of acquiredcancer stemness has not yet been described inhematological malignancies, although a good number ofCSC markers for these cancers have been proposed,including CD34+/CD38-/CD90−/interleukin 3 recep-tor(IL-3R)+/human leukocyte antigen (HLA)-DR/CD117-for acute myeloid leukemia (AML), CD34+/CD10-/CD19-/CD133+ for B-cell precursor acute lymphoblasticleukemia (ALL), and CD34+ for chronic myeloid leukemia(CML) [43, 44]. Regarding ALK+ALCL, we are aware of apublication in which tumor-propagating cells were identi-fied based on their high Hoechst-efflux ability [45]. Thesetumor-propagating cells are featured with higher expres-sion levels of NPM-ALK and ATP-binding cassette trans-porter G2 (ABCG2) as compared to bulk cell populations[45]. However, there was no evidence of a conversion ofnon-CSCs to CSCs in this study. Overall, we believe thatwe have demonstrated the first example of cancer cellplasticity in hematopoietic cancers.We have previously demonstrated the central role ofthe Wnt/β-catenin/MYC/Sox2 axis as the defining fea-ture of RR cells in ALK+ALCL [10]. Specifically, thehigh level of MYC permits the DNA binding of Sox2,and thus, promotes the transcription of Sox2 down-stream target genes. One of the downstream target genesis WNT2B, which in turn upregulates the Wnt canonicalpathway, resulting in an upregulation of MYC [10]. Thispositive feedback loop exists in RR cells but not RU cells[10]. In support of this model, we found that convertedRR cells induced by H2O2 showed upregulation of thissignaling axis. Compared to native RU cells, convertedRR cells were found to have a substantially high expres-sion level of Wnt2B, β-catenin, and MYC, as well as en-hanced Sox2-SRR2 binding. In contrast, inhibition of β-catenin, MYC, or Sox2 resulted in significantly deceasedconversion induced by oxidative stress. Although howexactly oxidative stress stimulates the Wnt/β-catenin/MYC/Sox2 axis is not known, redox oxidative speciesare known to directly activate the Wnt canonical path-way in HEK293 cells by stabilizing β-catenin and inter-rupting the interaction between dishevelled (Dvl) andnucleoredoxin (NRX), a strong inhibitor of Wnt/β-ca-tenin signaling [46]. Further studies are required to de-termine if these mechanisms indeed underlie the RU toRR conversion in ALK+ALCL cells challenged by oxida-tive stress.ConclusionsOur study has demonstrated a novel experimental modelin which the acquisition of CSC features can be inducedby oxidative stress in ALK+ALCL, a hematologic malig-nancy. Our results have further substantiated the import-ance of the Wnt/β-catenin/MYC/Sox2 axis in conferringthe cancer stem-like phenotype in ALK+ALCL.Additional filesAdditional file 1: Figure S1. The cell numbers of RU cells derived fromSupM2 and Karpas 299 upon various doses of H2O2 treatment werecounted by trypan blue exclusion assay from day 0 to day 3. (PDF 79 kb)Additional file 2: Figure S2. Anti-oxidant reagent NAC blocked the in-crease of GFP-positive cells induced by H2O2. A-B) Treatment of NAC ab-rogated the increased GFP-positive cells induced by 0.3 mM H2O2 for48 h in RU cells derived from SupM2 in a NAC-dose dependent manner,read by GFP expression (A) and luciferase activity (B). (PDF 55 kb)Additional file 3: Figure S3. The serial dilution experiment in RU andRR cells derived from Karpas 299 cells. The serial diluted RU and RR cellsderived from Karpas 299 (from 1000 cells to 1 cell) were seeded in 96-well plates. After 8 days, the number of spheres was counted in thehighlighted wells circulated by the rectangle lines. The right panelshowed the analyzed results which indicated that converted RR cells andnative RR cells with H2O2 stimulation have formed more spheres in alower number of cells seeded (125 cells for RU cells and converted RRcells, 32 cells for RR cells and RR cells with H2O2 stimulation), as com-pared with native RU and RR cells, respectively. Note that RR cells alsohave formed more spheres than RU cells at a lower number of cellsseeded (i.e. 32 and 63 cells). (PDF 259 kb)Additional file 4: Figure S4. The cell growth of RU and RR upon H2O2re-challenge. A-B) The cell growths of RU and RR cells derived fromSupM2 and Karpas 299 after H2O2 re-challenge, assessed from day 1 (day6 of H2O2 re-challenge experiment) to day 3. The results indicated thatconverted RR cells from both cell lines share similar cell growth rates withnative RU cells, and RR cells after H2O2 re-challenge also grow in a similarrate with native RR cells. (PDF 103 kb)Additional file 5: Figure S5. The activation levels of ALK and STAT3were inappreciably changed upon H2O2 re-challenge. The expressionlevels of pALKY1604, ALK, pSTAT3Y705, and STAT3 in RU and RR cells withor without H2O2 re-challenge. The same cell lysates from Fig. 3a werereused in this experiment, and note that the same β-actin blot as theone in Fig. 3a was recycled for H2O2-stimulation in RU and RR cells de-rived from Karpas 299 cells. (PDF 102 kb)Additional file 6: Figure S6. RU cells derived from SupM2 weretransfected with either Sox2 siRNA or scrambled siRNA which served as anegative control. Cells after siRNA transfection were exposed to 0.3 mMH2O2 re-challenge. At day 4 of the H2O2 re-challenge experiment; cellswere subjected to 200 ng/mL doxorubicin for additional 48 h, followingby the trypan blue exclusion assay-based cell viability analysis. TheWu et al. BMC Cancer  (2018) 18:361 Page 9 of 11Western blots in the right panel demonstrated the Sox2 knockdown effi-ciency in RU cells from SupM2 24 h post transfection. (PDF 48 kb)AbbreviationsALK+ALCL: ALK-positive anaplastic large-cell lymphoma; CSCs: cancer stemcells; GFP: Green Fluorescence Protein; H2O2: hydrogen peroxide; NAC: N-acetyl-L-cysteine; qRT-PCR: quantitative real-time polymerase chain reaction;RR: reporter responsiveness; RU: reporter unresponsiveness; siRNA: shortinterfering RNAAcknowledgementsWe thank Jingzhou Huang, the Core Flow Cytometry laboratory at theDepartment of Experimental Oncology, Cross Cancer Institute, University ofAlberta, for his technical favors.This article has been retrieved and modified from Wu C (2017). Delineationof molecular mechanisms underlying the pathobiology of ALK-positive ana-plastic large-cell lymphoma (Doctoral dissertation). Retrieved from https://era.library.ualberta.ca/files/cmc87pq51z/Wu_Chengsheng_201702_PhD.pdf.pdfFundingThis work was supported by an operating research grant from the CanadianInstitute of Health Research awarded to R.L. C.W. was a recipient of the theChina Scholarship Council scholarship. H.Z. was a recipient of the Li Ka Shingscholarship. The funding bodies did not play any role in the design of thisstudy, collection/analysis/interpretation of data and the writing ofmanuscript.Availability of data and materialsThe data supporting the findings of this study is available from thecorresponding author upon reasonable request.Authors’ contributionsCW and RL designed the experiments. CW performed most of theexperiments and participated in data interpretation. CW and RL wrote themanuscript. NG, YH, HZ, AA, and AC performed portions of the experiments,data analysis and intellectual input. NG and YH contributed equally to thisstudy. All authors read and approved the manuscript.Ethics approval and consent to participateN/AConsent for publicationN/ACompeting interestsThe authors declare that they have no competing interests.Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.Author details1Department of Laboratory Medicine and Pathology, University of Alberta,5142J Katz Group Centre for Pharmacy and Health Research, Edmonton, ABT6G 1Z2, Canada. 2Department of Oncology, University of Alberta,Edmonton, AB, Canada. 3DynaLIFEDX Medical Laboratories, Edmonton, AB,Canada. 4Department of Pathology and Laboratory Medicine, University ofBritish Columbia, Vancouver, BC, Canada. 5Department of LaboratoryMedicine and Pathology, Taibah University, Medina, Saudi Arabia. 6CurrentAddress: Department of Pathology, University of California San Diego, LaJolla, California, USA.Received: 5 May 2017 Accepted: 23 March 2018References1. 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Nat Cell Biol. 2006;8:501–8.•  We accept pre-submission inquiries •  Our selector tool helps you to find the most relevant journal•  We provide round the clock customer support •  Convenient online submission•  Thorough peer review•  Inclusion in PubMed and all major indexing services •  Maximum visibility for your researchSubmit your manuscript atwww.biomedcentral.com/submitSubmit your next manuscript to BioMed Central and we will help you at every step:Wu et al. BMC Cancer  (2018) 18:361 Page 11 of 11


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