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Published OnlineFirst April 9, 2015; DOI: 10.1158/0008-5472.CAN-14-2215

Cancer Tumor and Stem Cell Biology Research

Lin28B/Let-7 Regulates Expression of Oct4 and Sox2 and Reprograms Oral Squamous Cell Carcinoma Cells to a Stem-like State Chian-Shiu Chien1, Mong-Lien Wang2,3, Pen-Yuan Chu4,5, Yuh-Lih Chang2,6, Wei-Hsiu Liu7, Cheng-Chia Yu8, Yuan-Tzu Lan3,6, Pin-I. Huang3,9, Yi-Yen Lee3,9, Yi-Wei Chen3,9, Wen-Liang Lo1,10, and Shih-Hwa Chiou2,3,4,6,11

Abstract

Lin28, a key factor for cellular reprogramming and generation HMGA2 and suppressed their expression, whereas ARID3B and of induced pluripotent stem cell (iPSC), makes a critical con- HMGA2 increased the transcription of Oct4 and Sox2, respec- tribution to tumorigenicity by suppressing Let-7. However, it is tively, through promoter binding. Chromatin immunoprecipi- unclear whether Lin28 is involved in regulating cancer stem–like tation assays revealed a direct association between ARID3B and cells (CSC), including in oral squamous carcinoma cells aspecific ARID3B-binding sequence in the Oct4 promoter. (OSCC). In this study, we demonstrate a correlation between Notably, by modulating Oct4/Sox2 expression, the Lin28B–Let7 high levels of Lin28B, Oct4, and Sox2, and a high percentage of pathway not only regulated stemness properties in OSCC but þ þ CD44 ALDH1 CSC in OSCC. Ectopic Lin28B expression also determined the efficiency by which normal human oral in CD44 ALDH1 /OSCC cells was sufficient to enhance keratinocytes could be reprogrammed to iPSC. Clinically, a Oct4/Sox2 expression and CSC properties, whereas Let7 co- Lin28Bhigh-Let7low expression pattern was highly correlated with overexpression effectively reversed these phenomena. We iden- high levels of ARID3B, HMGA2, OCT4, and SOX2 expression in tified ARID3B and HMGA2 as downstream effectors of Lin28B/ OSCC specimens. Taken together, our results show how Lin28B/ Let7 signaling in regulating endogenous Oct4 and Sox2 expres- Let7 regulates key cancer stem–like properties in oral squamous sion. Let7 targeted the 30 untranslated region of ARID3B and cancers. Cancer Res; 75(12); 2553–65. 2015 AACR.

Introduction within months after current therapeutic treatments and around 50% of patients die of this disease or die from complications Head and neck squamous cell carcinoma (HNSCC), includ- within 5 years under current therapies. Embryonic stem cell ing oral squamous cell carcinoma (OSCC), is the sixth most (ESC) signatures, including Oct4, Sox2, Nanog, Klf4, and prevalent malignancy worldwide (1, 2). Most patients relapse c-Myc, have been reported to maintain the self-renewal property and drive cellular reprogramming into pluripotent state 1Institute of Oral Biology, National Yang-Ming University, Taipei, in normal somatic cell as well as in malignant transformed 2 Taiwan. Institute of Pharmacology, National Yang-Ming University, cells. Exogenous induction of ESC-stemness gene has been Taipei, Taiwan. 3Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan. 4School of Medicine, National Yang-Ming shown to promote dysplastic growth in adult epithelial tissues University, Taipei, Taiwan. 5Laryngology and Head and Neck Surgery, (3, 4). A recent study indicated that premature termination Department of Otolaryngology, Taipei Veterans General Hospital, Tai- of reprogramming may produce cells that are equipped with pei,Taiwan. 6Department of Medical Research,TaipeiVeterans General Hospital, Taipei, Taiwan. 7Graduate Institute of Medical Sciences, several stemness properties but fail to become successfully National Defense Medical Center and Tri-Service General Hospital, induced pluripotent stem cell (iPSC), and finally generate Taipei, Taiwan. 8Institute of Oral Science, School of Dentistry & Oral neoplasia resembling Wilms tumor (5). These interesting find- Medicine Research Center, Chung Shan Medical University, Taipei, Taiwan. 9Cancer Center, Taipei Veterans General Hospital, Taipei, ings suggested a possible link between ESC/stemness signature- Taiwan. 10Division of Oral and Maxillofacial Surgery, Department of mediated reprogramming and tumor transformation. Stomatology, Taipei Veterans General Hospital, Taipei, Taiwan. 11Geno- Lin28 has been shown to be a key RNA-binding protein and mics Research Center, Academia Sinica, Taipei, Taiwan. plays a critical role in regulating the balance between stemness Note: Supplementary data for this article are available at Cancer Research and differentiation in ESCs (6). Lin28 is highly expressed during Online (http://cancerres.aacrjournals.org/). embryogenesis and has emerged as a factor that defines stemness C.-S. Chien, M.-L. Wang, and Y.-L. Chang contributed equally to this article. state in several tissue lineages (7). Lin28 was found to be a master Corresponding Authors: Shih-Hwa Chiou, Department of Medical Research, regulator of developmentally timed changes in hematopoietic Taipei Veterans General Hospital, No. 201, Sec. 2, Shih-Pai Road, Taipei 11217, stem cells (HSC) programs with the high-mobility group AT-hook Taiwan. Phone: 886-2-28757394; Fax: 886-2-28757435; E-mail: 2 (HMGA2) serving as its specific downstream modulator of [email protected]; and Wen-Liang Lo, Division of Oral and Maxillofacial HSC self-renewal potential (8). Overexpression of Lin28 was Surgery, Department of Stomatology, Taipei Veterans General Hospital, Taipei, detected in various cancers and involved in the oncogenesis of Taiwan. E-mail: [email protected] human malignancies (9–12). Moreover, through inhibiting doi: 10.1158/0008-5472.CAN-14-2215 Let7 biogenesis, Lin28 influences mRNA translation, regulates 2015 American Association for Cancer Research. the self-renewal of ESCs, and promotes the malignant

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transformation of cancers (8, 13–15). Recent reports showed that evaluation of cell proliferation, cells were seeded in 24-well the Lin28–Let7 pathway plays a critical role in regulating the plates at a density of 2 104 cells per well in culture medium stemness, self-renewal, tumor initiation, and epithelial–mesen- and subjected to methyl thiazole tetrazolium assay (MTT assay; chymal transition–derived metastasis in malignant tumors and Sigma-Aldrich Co.). cancer stem–like cells (CSC; refs. 8, 9, 12, 13, 15, 16). However, the detailed molecular mechanism involved in the Lin28/Let7– Human iPSC generation and culture driven stemness regulation and cancer reprogramming is still an Normal human oral keratocyte (NHOK) primary cells (21) open question. were reprogrammed by the transcription factors of Oct4, Sox2, Oct4 is a key reprogramming factor and balances the pluri- Klf4, and c-Myc (OSKM) or a combination of Oct4, Sox2, potent and differentiated states in stem cell and cancer devel- Nanog, and Lin28 (OSNL) using a retroviral transfection opment (4, 17, 18). Sox2 is critical in maintaining self-renewal (23, 24). In briefly, the retroviral-based system carrying the of ESC (17), and Sox2-mediated pathway was also shown to four transcription factors (OSKM or OSNL) was generated by regulate the acquisition of CSC-like and radiochemoresistant transfecting 10 mg of each transcription factor-encoding pMXs properties in HNSCC (19). A genomic copy number gain of the plasmid in 2.5 106 Plat-A cells using TransIT-LT1 (Mirus). Sox2 locus was reported in OSCC, which resulted in an increase The retroviral-containing supernatants were collected and fil- of Sox2 transcriptional activity and might be critically involved tered through 0.45-mm filters 48 hours after transfection, and in OSCC initiation and progression (20). Although Oct4 and equal amounts of each of the four retroviral supernatants Sox2 are seemingly involved in the induction of CSC properties, supplemented with 10 mg/mL polybrene (Sigma) were applied the regulatory mechanisms of ESC/stemness signature-depen- on 5 104 target cells in 6-well plates. The 6-well plates were dent modulation of CSC properties in OSCC are still blurred. centrifuged at 2,250 rpm for 1 hour, and the medium was Here, we reported that the Lin28B–Let7 pathway positively replaced. On day 7 of postinfection, reprogramming cells regulates Oct4 and Sox2 expression, inducing a reprogram- were passaged onto mitotically inactivated mouse embryonic ming-like phenomenon, switching non-CSCs to CSCs with fibroblast (MEF) feeder layers, and cultured using human ESC tumor-initiating and self-renewal properties in oral CSC. We medium [DMEM/F12 (Gibco) supplemented with 20% knock- identified the AT-rich interaction domain molecule 3B out serum replacer (KSR; Invitrogen), 0.1 mmol/L nonessential (ARID3B) and HMGA2 as direct targets of Let7, mediating Oct4 amino acids (Invitrogen), 1 mmol/L L-glutamine, 0.1 mmol/L and Sox2 expression by direct regulation of Oct4 and Sox2 b-mercaptoethanol, 10 ng/mL recombinant human bFGF, and promoter activity, respectively. These data suggested that the antibiotics (Gibco)]. SB431542 (2 mmol/L; Stemgent), Lin28B–Let7pathwayservesasadrivermechanismtopromote PD0325901 (0.5 mmol/L; Stemgent), and thiazovivin ESC-stemness properties through inducing a reprogramming- (0.5 mmol/L) were added to the culture medium to aid colony like mechanism in OSCC cells. formation and this medium was refreshed daily until iPSC colonies appeared (25). Undifferentiated iPSCs were main- tained on mitotically inactivated MEFs (50,000 cells/cm2)in Materials and Methods human ESC medium. To prevent MEF contamination, human þ þ Cell culture, CD44 ALDH1 OSCC cells isolation, and tumor iPSCs were transferred to a feeder-free/serum-free CSTI-8 medi- sphere enrichment um (Cell Science & Technology Institute Inc.) without KSR All procedures of tissues acquirements have followed the supplementation. The detailed information for generating and tenets of the Declaration of Helsinki and are approved by the long-term maintaining iPSCs has been described in our previ- Institutional Ethics Committee/Institutional Review Board of ous report (23, 24). Taipei Veterans General Hospital and Chuang Sang Hospital (Taipei, Taiwan). All samples were obtained after patients pro- SPONGE constructions vided informed consent. All culture protocols and information of Let7-SPONGE, scramble, and antisense Let7 and microRNA OSCC cell isolated from patients were followed our previous were constructed using a pcDNA 6.2-GW/EmGFP-miR plasmid reports (21). Within 1 hour after surgical removal, tumors were (Invitrogen; ref. 26). microRNA SPONGE sequence design was washed and enzymatically dissociated into single cells. The dis- based on Ebert and colleagues (27), and the sequence of Let7- sociated cells derived from the samples of OSCC patients were SPONGE was listed in Supplementary Table S3. suspended and cultured at a concentration of 1 106/mL in 37C DMEM with 2% FCS. To identify CD44-positive and ALDH1- Bisulfite pyrosequencing positive cells, we stained cells with CD44 antibody (phyco- Bisulfate treatment and DNA cleanup was performed by using erythrin-conjugated; BioLegend) and applied Aldefluor assay the EpiTect 96 Bisulfite Kit according to the previous protocol (STEMCELL Technologies) followed by fluorescence-activated (23, 26). In brief, 500 ng of genomic DNAs were reacted with cell sorting analysis (FACS; refs. 21, 22). For enrichment of freshly prepared bisulfite/hydrochinone solutions to convert spheres, 103 cells were plated in low-attachment 6-well plate unmethylated cytosine to uracil, and methylated cytosine was (Corning Inc.) with tumor sphere medium consisting of protected from the chemical reaction. The bisulfite reaction serum-free DMEM/F12 medium (GIBCO), N2 supplement was stopped by neutralization using sodium acetate, and the (GIBCO), 10 ng/mL human recombinant basic fibroblast growth genomic DNAs were precipitated with glycogen by ethanol DNA factor (bFGF) and 10 ng/mL epidermal growth factor (EGF; R&D precipitation method. PCR amplification primers and sequen- Systems). For serial passage of spheroid cells, single cells were cing primers were designed by PyroMark Assay Design Software obtained from Accutase (STEMCELL Technologies)–treated 2.0. PCR amplification of target region was performed by using spheroids and the cells were seeded at a density of 1,000 cells/mL the PyroMark PCR Kit (Qiagen). Details are described in the in the serum-free medium as described above (21, 22). For Supplementary Data.

2554 Cancer Res; 75(12) June 15, 2015 Cancer Research

Lin28/Let-7 Reprogramming OSCC into Stem-like State

Let7 angomir and liposome-mediated delivery with the parental cells and empty vector-transfected controls Let7 RNA oligos were synthesized as identical sequences of (Fig. 1G and H; Supplementary Fig. S1A). Let7 with modifications. The phosphodiester backbones were Functional analysis showed that CD44 ALDH1 /pLV- modified by phosphothiolate backbones and the ribose was Lin28B cells presented enhanced sphere formation capability replaced by lock-nucleic-acid (LNA) ribose. The delivery of the in comparison with control cells; whereas knockdown of þ þ þ þ Let7 angomir was mediated by liposome-based nucleic acid Lin28B in CD44 ALDH1 cells (CD44 ALDH1 /shLin28B) delivery method. In brief, Let7 was dissolved in D5W (5% suppressed the formation of suspended spheres (Supplemen- dextrose water) and packaged with specialized lipid compo- tary Fig. S2A) and soft agar colonies (Supplementary Fig. S2B). nents into liposomes. The packaged liposomes (final concen- Co-knockdown of Oct4 and Sox2 by short hairpin RNA tration of Let7 angomir is 10 ng/mL) were delivered into the (shRNA) in CD44 ALDH1 /pLV-Lin28B cells (Fig. 1H) same locus of intracranially xenotransplanted tumor-initiation decreased the sphere number (Fig.1I),indicatingaroleof cells. Lin28B in regulating self-renewal and tumorigenesis in OSCC cells. In addition, CD98 was recently shown as a marker to Statistical analysis enrich a subpopulation of HNSCC cells with stem cell pro- Data are presented as mean SD. A Student t test or analysis of perties (29). However, our flow cytometry data showed no variance (ANOVA) test was used to compare the continuous significant difference of CD98 level between the functionally þ þ variables between groups, as appropriate. The x2 test or Fisher proved CSC-like CD44 ALDH1 /shCtrl and non-CSC-like þ þ exact test was used to compare the categorical variables. P < 0.05 CD44 ALDH1 /shLin28 cells (data not shown). Finally, trans- was considered statistically significant. plantation of these stable cells in the neck region of immunocompromised mice showed that overexpression of Lin28B increased tumor growth in CD44 ALDH1 cells, whereas Results co-knockdown of Oct4 and/or Sox2 (CD44 ALDH1 /pLV- Elevated Lin28B in high-grade and CSC-like OSCC cells were Lin28B/shOct4þshSox2)decreasedit(Fig.1J).Notably,the correlated with increased Oct4, Sox2, and sphere formation Oct4 and Sox2 seemed to have an additive effect in this assay, as Lin28isrecentlyafocusinthefield of regenerative researches knockdown of both presented a better suppressive effect on as its role in RNA regulation has been linked to the modi- tumorgrowththanknockdownofeach(Fig.1J).Consistently, fication of miRNA landscape during ESCs differentiation as subcutaneous transplantation of these stable cell lines in the well as somatic cell reprograming. Although stemness factors, neck region of immunocompromised mice with different cell such as Oct4 and Sox2, have been implicated in malignant numbers indicated that Lin28B overexpressed cells were able development and stemness property acquisition of cancers, to induce tumor formation with as less as 1,000 cells, whereas the role of Lin28B in cancer stemness, however, is still unclear. the control cell line could not generate in vivo tumors even with We first analyzed and compared the expression levels of Lin28B a cell number up to 100,000 (Supplementary Fig. S1E). in nine pairs of tumorous and nontumorous tissues of oral Co-knockdown of Oct4 and Sox2 in the CD44 ALDH1 / cancer patients. Immunohistochemistry (Fig. 1A) and quanti- pLV-Lin28B cells hampered the tumor-initiation ability in vivo tative real-time PCR (qRT-PCR; Fig. 1B) showed that Lin28B (Supplementary Fig. S1E). These data indicated that Lin28B was highly expressed, both in protein and mRNA levels, in oral induced stemness genes expression such as Oct4 and Sox2, CSC cancer specimens, compared with the nontumorous counter- markers expression such as CD44 and ALDH1, self-renewal parts. Further analysis of Lin28B levels in different stages of potential, and tumor growth and initiation. A novel Lin28B- OSCC specimens revealed a correlation between high Lin28B Oct4–Sox2 pathway was identified in regulating stemness levels and advanced stage of OSCC (Fig. 1C). property of OSCC. OSCC cells that express CD44 and ALDH1, and are able to form spheres in suspension culture, were proposed to be CSCs Let7 is a negative downstream effector of Lin28B in the with tumor-initiating potential (28). These markers, therefore, regulation of Oct4/Sox2 and tumorigenesis in OSCC were used to identify OSCC-CSCs. Using flow cytometry, we Let7 is a negative downstream molecule of Lin28 (8, 14, 15); þ þ isolated CD44 and ALDH1 cells from 3 patients' tumorous Lin28B-mediated inhibition of Let7 resulted in upregulated specimens (Pt1-3) and found that cells expressing these CSC self-renewal and epithelial–mesenchymal transition in HSCs markers had high mRNA expression levels of Lin28B (Fig. 1D). and cancers (8, 14, 15). We, therefore, investigate whether Let7 Serial transfection of the isolated cells in NOD/SCID mice is one of the major downstream effectors of Lin28B in regu- showed significant higher tumor-initiating potential of lating CSC-like and reprogramming property in OSCC. We first þ þ CD44 ALDH1 cells than CD44 ALDH1 cells (Supplemen- stably suppressed the level of Let7 in patient-derived CD44 tary Fig. S1E). We then exogenously overexpressed Lin28B in ALDH1 cells by knockdown of Let7 expression using the three patient-derived CD44 ALDH1 cells (CD44 ALDH1 / SPONGE system. SPONGE is a competitive inhibitor of miRNA pLV-Lin28B; Fig. 1H; Supplementary Fig. S1A) and showed that through strong binding with the miRNA of interest by its þ overexpressed Lin28B increased the percentage of CD44 multiple binding sites, relieving the targets of the miRNA from þ ALDH1 cells (Fig. 1E) as well as the sphere-forming ability being inhibited (27). We established Let7 SPONGE, a compet- (Supplementary Fig. S1B). Moreover, cells expressing CD44 itive inhibitor containing a transcript expressing multiple, and ALDH1, individually or simultaneously, also presented tandem binding sites to Let7. When vectors encoding Let7 high level of Oct4 and Sox2, compared with their nonexpres- SPONGE are expressed in cells, the SPONGE strongly competes sing counterparts (Fig. 1F; Supplementary Fig. S1C and S1D). with the targets of Let7, "absorbs" Let7 in cells, and relieves Similarly, in the CD44 ALDH1 /pLV-Lin28B cells, Oct4 and the targets of Let7 from being suppressed by Let7. In this Sox2 were increased in mRNA and protein levels, compared study, we used Let7 SPONGE (Spg-Let7) as a knockdown

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A BF HE Lin28B P = 0.028 – – CD44 /ALDH CD44–/ALDH– 4 + + 8 + + 15 CD44 /ALDH CD44 /ALDH 3 6 10 2 4 Nontumor 1 2 5

Fold change of Sox2 0 Fold change of Oct4 0 Pt 1 Pt 2 Pt 3 Pt 1 Pt 2 Pt 3 Lin28B (2- D CT)

Expression level of Expression level 0 G pLV-Ctrl pLV-Ctrl Tumor Nontumor Tumor 20 pLV-Lin28B 20 pLV-Lin28B N = 9 N = 9 C 15 15 40 10 10 30 5 5 Fold change of Oct4 20 0 Fold change of Sox2 0 Pt 1 Pt 2 Pt 3 Pt 1 Pt 2 Pt 3 10 Pt 1 CD44–/ ALDH1– H pLV-Ctrl

Lin28B (2- D CT) + −−−− pLV-Lin28B − ++++ Expression level of Expression level 0 I II III IV ShOct4 − − + − + ShSox2 − − − + + D Lin28B – – 3.5% CD44 /ALDH CD44+/ALDH+ Oct4

Sox2

GAPDH Pt 1 CD44–/ALDH1– + Lin28B I J Pt 1 CD44–/ALDH1– 0.1% Lin28B

Fold change of Lin28B Lin28B + ShSox2 Pt 1 Pt 2 Pt 3 pLV-Ctrl 2.0 Lin28B + ShOct4 E pLV-Ctrl pLV-Lin28B 1.8 Lin28B + ShOct4 + ShSox2 1.6 pLV-Ctrl 0.1% 0.1% 25 pLV-Ctrl 1.4 ShOct4 + /Shsox2

BEAB pLV-Lin28B 1.2 + 20 Lin28B + ShCtrl Lin28B + ShOct4 + ShSox2 1.0

IgG 60

/ALDH 0.8 + 15 50 0.6 10 40 2.6% 15.7% 30 (cm) volume Tumor 0.4

BAAA 0.2 + 5 20

Spheres ( n ) 10 0.0 % of CD44 0 0 0123456 CD44 Pt 1 Pt 2 Pt 3 Pt 1 Pt 2 Pt 3 Weeks

Figure 1. Elevated Lin28B in high-grade and CSC-like oral cancer cells was correlated with increased Oct4, Sox2, and sphere formation. A and B, nine pairs of tumor and nontumor specimens were derived from OSCC patients and subjected to IHC and qPCR analysis to assess the Lin28B protein and mRNA levels. One representative IHC stain of Lin28B is shown in A. C, samples from patients with different stages (stage I to IV) of OSCC were collected and subjected to þ þ qPCR for Lin28B mRNA level. D, primary culture cells derived from patients were subjected to sort out CD44 (left) and ALDH1 cells by using flow cytometry. þ þ qPCR was applied to analyze the Lin28B mRNA levels in CD44 ALDH1 and CD44 ALDH1 cells. Relative Lin28B expression level is shown in the quantitative chart on the right. E, CD44ALDH1 cells were transfected with pLV-Lin28B or empty vector (pLV-Ctrl) using lentiviral system. Both cells were subjected to flow cytometry analysis for their CD44 expression and ALDH1 activity. F, CD44þALDH1þ and CD44ALDH1 cells were subjected to qPCR analysis to assess the Oct4 and Sox2 mRNA expression. G, pLV-Ctrl and pLV-Lin28B-transfected cells were analyzed by qPCR for the Oct4 and Sox2 expression. H, patient-derived CD44ALDH1 cells with stably transfected plasmid constructs as indicated were subjected to Western blot analysis for the protein expression levels of Lin28, Oct4, and Sox2. I, pLV-Lin28B cells with or without double knockdown of Oct4 and Sox2 were subjected to a sphere formation assay. The results of tumorsphere formation by phase contrast microscopy are presented at the top and the quantified sphere number is presented in the graph at the bottom. This experiment was repeated in three patient-derived CD44 ALDH1 cells. , P < 0.05 versus control. Scale bar, 100 mm. J, stable cell lines as indicated were transplanted in the neck subcutaneous region of immunocompromised mice (n ¼ 3; each mouse was injected by 106 cells). Tumor volumes (mean tumor volume cm3 SEM) were measured by a caliper and monitored up to 6 weeks.

method of Let7, and validate the hypothesis of Let7 as a key expression of Let7 (Fig. 2B). Delivery of Let7-mimic had the mediator linking stemness genes with Lin28. In line with our same effect (Supplementary Fig. S2C and S2D). Sphere forma- hypothesis, suppressing Let7 expression in CD44 ALDH1 tion (Fig. 2C, left) and soft agar (Fig. 2C, right) assays indicated þ þ cells, which express higher level of Let7 than CD44 ALDH1 that delivery of Let7-mimic reversed the Lin28B-increased cells, resulted in increased Oct4 and Sox2 protein levels (Fig. sphere and colony numbers, resulting in a decrease of sphere 2A). We then overexpressed Let7 in CD44 ALDH1 /pLV- formation ability and anchorage independence down to a level Lin28B cells to generate a stable cell line (CD44 ALDH1 / similar to CD44 ALDH1 /pLV-Ctrl cells. pLV-Lin28BþLet7). An empty vector-transfected control cell To assess the effects of the Lin28B–Let7 pathway in tumor þ þ line was also established simultaneously (CD44 ALDH1 / initiation and growth, we established a panel of CD44 ALDH1 pLV-Lin28BþVec).WehaveshownthattheLin28B-elevated stable cell lines with Lin28B and Let7 being knocked down by protein levels of Oct4 and Sox2 were decreased upon co-over- shRNA and Sponge (26), respectively (Supplementary Fig. S2E).

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Lin28/Let-7 Reprogramming OSCC into Stem-like State

A Pt 1 CD44–/ALDH1– Pt 2 CD44–/ALDH1– D D5 D7 D14 D21 − − Spg-Ctrl − + − + 100 Pt 1

Spg-Let7 − − + − − + m Pt 2 μ ShCtrl Pt 3 Oct-4 80 50 Sox-2 > 60

GAPDH ShLin28B 40 20 B PT 1 PT 2 % of sphere > ShLin28B pLV-Ctrl pLV-Ctrl + Spg-Ctrl 0 + − − + − − ShCtrl + −−− pLV-Lin28B − ++ pLV-Lin28B − ++ ShLin28B − +++ pLV-Let7 − − + pLV-Let7 − − + Spg-Ctrl − −−+ Spg-Let7 − −−+ ShLin28B Lin28B Lin28B + Spg-Let7 Oct 4 Sox2 Oct-4 Oct-4 E 1.5 Pt 1 1.5 Pt 1 Pt 2 Pt 2 Sox-2 Sox-2 Pt 3 Pt 3 1.0 1.0 GAPDH GAPDH

mRNA 0.5 mRNA 0.5 8 16 4 8 Fold change of Fold change of 4 0.0 0.0 2 2 1 1 ShCtrl + −− − ShCtrl + −− −

of Let-7 0.5 of Let-7 fold change fold change fold 0.5 ShLin28B ++ + ShLin28B +++ 2 2 − − 0.25 0.25 + Spg-Ctrl + 0.125 0.125 Spg-Ctrl − −− − −− Log Log Spg-Let7 − −− + Spg-Let7 − −− +

C CD44–/ALDH1– CD44–/ALDH1– CD44+/ALDH1+ F G CD44+/ALDH1+ Pt 1 ShCtrl Injected 80 125 Pt 1 1.6 ShLin28B ShLin28B + Pt 2 Pt 2 ShLin28B + Spg-Let7 cell ShCtrl ShLin28B Pt 3 ShLin28B + Spg-Ctrl + Spg-Ctrl Spg-Let7 70 Pt 3 1.4 number 100 ShLin28B )

60 3 1.2 No.1 100,000 3/3 1/3 0/3 2/3 50 75 10,000 3/3 0/3 0/3 2/3 40 1.0 1,000 3/3 0/3 0/3 1/3 30 50 0.8

Spheres ( n ) No.2 100,000 3/3 1/3 1/3 2/3 Colonies ( n ) 20 0.6 25 10,000 3/3 0/3 0/3 1/3 10 0.4 1,000 3/3 0/3 0/3 1/3

0 0 (cm volume Tumor No.3 100,000 3/3 0/3 1/3 2/3 pLV-Ctrl 0.2 pLV-Ctrl 10,000 3/3 0/3 0/3 2/3 0.0 1,000 2/3 0/3 0/3 1/3 pLV-Lin28BpLV-Lin28BpLV-Lim28B pLV-Lin28BpLV-Lin28BpLV-Lim28B 123456 + Mimic-ctrl + Mimic-ctrl + Let7 mimic + Let7 mimic Weeks

Figure 2. Let7 is a negative downstream effector of Lin28B in the regulation of Oct4/Sox2 and tumorigenesis in OSCC. A, Oct4 and Sox2 expression in CD44 ALDH1 cells transfected with or without Spong-Let7 (Spg-Let7) and Sponge-control (Spg-Ctrl) was assessed by Western blot analysis. B, pLV-Ctrl, pLV-Lin28B, and pLV-Let7–transfected CD44ALDH1 stable cells were subjected to Western blot analysis for Lin28B, Oct4, and Sox2 expression (top) and qPCR for Let7 levels (bottom). C, CD44ALDH1 cells stably transfected with pLV-Ctrl, pLV-Lin28B, scrambled miR-mimic control, and Let7-mimic were subjected to sphere formation (left) and soft-agar (right) assay. The numbers of sphere and colony were calculated and are presented in the graphs. , P < 0.05 versus control. #, P < 0.05 versus Spg. D, CD44þALDH1þ cells with or without knockdown of Lin28B (shLin28B) and Let7 (Spg-Let7) were subjected to a sphere formation assay for up to 21 days. The size of sphere from each stable cell line is presented by phase contrast microscopy on the left, and the þ þ quantitative sphere number is presented in the graph on the right. Only spheres with a diameter more than 50 mm were counted. E, CD44 ALDH1 þ þ cells with or without knockdown of Lin28B and Let7 were subjected to qPCR to assess the expression level of Oct4 and Sox2. F, CD44 ALDH1 stable cell lines were transplanted in immunocompromised mice and the xenograft tumor volume was monitored up to 6 weeks. G, CD44þALDH1þ stable cell lines as indicated were subcutaneously transplanted in the neck region of immunocompromised mice with different cell number from 1,000 to 100,000 cells per transplantation. Each cell line was repeated in three mice, and the tumor formation was monitored for at least 3 months. The number of mice that presented visualized tumor mass is listed in the table.

Consistent with previous results, shLin28B suppressed the sphere ARID3B and HMGA2 are targets of Lin28B/Let7 signaling to formation (Fig. 2D) and Oct4 and Sox2 expression (Fig. 2E; regulate Oct4 and Sox2 Supplementary Fig. S2E); whereas Sponge-Let7 (Spg-Let7) Following the finding of the Lin28B–Let7–Oct4/Sox2 path- reversed these effects (Fig. 2D and E). We then transplanted these way, we then analyzed the direct regulatory mechanism of how stable cells lines in the neck subcutaneous region of immuno- Lin28B mediates the expression of the two important stemness þ compromised mice with the indicated number of cells. In CD44 factors. We applied a bioinformatics analysis in three pairs of þ þ þ ALDH1 cells, shLin28B suppressed the tumor growth and ini- isolated OSCC cells: CD44 ALDH1 versus CD44 ALDH1 , tiation, both of which were rescued by Spg-Let7 (Fig. 2F and G). CD44 ALDH1 /Ctrl versus CD44 ALDH1 /Lin28B, and prima- These results demonstrated that Let7 is the key downstream ry culture of NHOK versus NHOK-iPSC, and compared their effectors of Lin28B, negatively mediating Lin28B-dependent reg- gene expression profiles. We identified ARID3B and HMGA2 ulation of tumorigenesis and stemness-derived reprogramming as potential regulators (Fig. 3A). ARID3B is a DNA-binding properties in OSCC. protein belonging to the AT-rich interactive domain (ARID)

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A CE Pt 1 CD44+/ALDH1+ Pt 1 CD44–/ALDH1–

ShLin28B Spg-Let7 ShCtrl Spg-Ctrl pLV-Ctrl + + −− ShCtrl + + −− HMGA2 − −−+ ShHMGA2 − −−+ Sox2 − − − + ShSox2 − − − +

HMGA2 HMGA2

ARID3B ARID3B

Sox2 Sox2

Oct4 Oct4 HMGA2, Sox2, ARID3B and Oct4...etc GAPDH GAPDH

B Pt 1 CD44–/ALDH1– D Pt 1 CD44+/ALDH1+ F Pt 1 CD44–/ALDH1– pLV-Ctrl + −−−−− pLV-Lin28B − ++++ − ShLin28B Spg-Let7 ShCtrl Spg-Ctrl pLV-Let7 − − + + − − pLV-Ctrl + + −− ShCtrl + + −− Spg-Ctrl − − − + − − ARID3B − −−+ ShARID3B − −−+ Spg-Let7 − − −−++ HESC-S6 Human iPSC Oct4 − − − + ShOct4 − − − + HMGA2 HMGA2 HMGA2 ARID3B ARID3B ARID3B

Oct4 Sox2 Sox2

Oct4 Oct4 Sox2

GAPDH GAPDH GAPDH

Figure 3. The Lin28B-Let7-Oct4–Sox2 pathway mediated the stemness and tumorigenesis properties in OSCC. A, three pairs of OSCC cells, CD44þALDH1þ versus CD44ALDH1,CD44ALDH1/Ctrl versus CD44ALDH1/Lin28B, and the primary culture of NHOK versus NHOK-iPSC were subjected to microarray and bioinformatics analysis to compare their gene expression proﬁles. The consistently changed molecules in the three pairs of cells were selected as potential downstream factors of the Lin28B–Let7 pathway in OSCC tumorigenesis and reprogramming process. In our data, we identiﬁed ARID3B, HMGA2, Oct4, and Sox2 as subjects for further investigations. B, CD44 ALDH1 OSCC cells with different combinations of overexpressed Lin28B, Let7, and Spg-Let7 were analyzed by Western blot analysis for the protein expression levels of ARID3B, HMGA2, Oct4, and Sox2. The human ES cells (HESC-S6, human ESC line) and human iPSC were the positive control for the four molecules in the blot. C and D, CD44þALDH1þ/shLin28B cells were transfected with or without ARID3B, HMGA2, Oct4, and Sox2 before being subjected to Western blot analysis. E and F, CD44ALDH1/Spg-Let7 cells with or without knockdown ofARID3B,HMGA2,Oct4,andSox2wereanalyzedbyWesternblotanalysis.

þ þ family of transcriptions factors that are involved in diverse bio- sing the four molecules in CD44 ALDH1 /shLin28B cell line. logic processes (30), and HMGA2 is a member of the high- Overexpression of ARID3B and HMGA2 enhanced the protein mobility group AT-hook protein family and has been reported levels of Oct4 and Sox2, respectively, whereas overexpression to be a Let7 target (8) and directly binds to Sox2 promoter in of Oct4 and Sox2 did not affect the levels of ARID3B and glioblastoma cells to induce Sox2 expression (26). To validate the HMGA2(Fig.3CandD).KnockdownofARID3BandHMGA2 bioinformatics data, we established a panel of patient sample- in CD44 ALDH1 /Spg-Let7 cells resulted in suppressed Oct4 derived CD44 ALDH1 OSCC cells with different combination and Sox2, respectively, but not vice versa (Fig. 3E and F). Taken of overexpressed Lin28B, Let7, and Spg-Let7. We found that together, these results demonstrated the causal effect of Lin28B/ overexpressed Lin28B increased the protein levels of ARID3B, Let7–driven ARID3B–HMGA2-Oct4–Sox2 pathway in OSCC. HMGA2, Oct4, and Sox2, whereas co-overexpression of Let7 reversed this effect of Lin28B and resulted in suppressed protein Let7 regulates tumor initiation of OSCC through targeting the levels of the four molecules down to a level similar to control cells 30-UTR of ARID3B and HMGA2 (Fig. 3B). Spg-Let7 dramatically increased the expression of the Using the TargetScan program, we identiﬁed potential Let7- four molecules, and this increase was even more signiﬁcant with targeting sites in the 30-untranslated region (30-UTR) regions of co-overexpression of Lin28B (Fig. 3B). ARID3B. We constructed reporter plasmids containing either Moreover, we dissected the regulatory causal effects between full-length, serial deletion (D1-D4, the potential Let7 targeting ARID3B, HMGA2, Oct4, and Sox2 by individually overexpres- site was deleted), or mutated forms of the 30-UTR region of

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Lin28/Let-7 Reprogramming OSCC into Stem-like State

AD Pt 1 CD44+/ALDH1+ 2325 2348 1.25 Arid3B 3’UTR hsa let-7 5′-UAUUUCAAUAAACCUCUACCUCU-3′ 1.00 0.75 Full length Luciferase 2369 Parental pLV-Ctrl pLV-Let-7 0.50 D1 Luciferase 1950 ARID3B 0.25 Fold change D2 Luciferase 1500 0.00

D3 Luciferase 1000 GADPH Parental pLV-Ctrl pLV-Let-7 D4 Luciferase 250

Mut Luciferase 2369 E Pt 1 CD44–/ALDH1–

hsa let-7 5′-UAUUUGCAUAAACCUAAACAUCA-3′ 7.5 2325 2348 B 5.0 Parental Spg-Ctrl Spg-Let7 2325 2348 2.5 ′ ′ ARID3B ARID3B 3’UTR 5 –UAUUUCAAUAAACCU–CUACCUCU–3 Fold change 0.0 ′ ′ hsa let-7 3 –UUGGUGUGUUGGAUGAUGGAGU–5 GADPH ′ ′ Mut ARID3B 3’UTR 5 –UAUUUGCAUAAACCU–AAACAUCA–3 Parental Spg-Ctrl Spg-Let7 F C CD44–/ALDH1– 1.2 Spg-Let7 125 pLV-Ctrl 125 pLV-Ctrl pLV-Let-7 pLV-Let-7 + Let7 antisense 1.0 Spg-Let7 + ShARID3B 100 100 Parental 0.8 pLV-Ctrl 75 75 0.6 50 50 0.4 Fold change of

25 Fold change of 25 relative activities (%) relative activities (%)

Tumor volume (cm) volume Tumor 0.2 0 0 Full D1 D2 D3 D4 Mut Full 0.0 length D1 D2 D3 D4 Mut length 123456 Weeks

Figure 4. ARID3B is a direct target of Let7 pathway in controlling Oct4 expression and tumorigenesis. A, schematic presentation of the constructed ARID3B 30-UTR reporter plasmids used in this study. The potential Let7-binding site is presented as a black box in full-length reporter plasmid. B, presentation of the sequence of wild-type ARID3B 30-UTR, Let7, and generated mutations in ARID3B 30-UTR reporter plasmid. C, the ARID3B 30-UTR reporter plasmids þ þ were cotransfected with control vector or Let7-expressing plasmid (left)/Let7-expressing plasmid with Let7 antisense (right) in CD44 ALDH1 OSCC cells. The luciferase activity of each combination was quantiﬁed and is presented in the graph. D, Western blot (left) and qPCR (right) analysis of CD44þALDH1þ cells with or without overexpression of Let7. E, Western blot (left) and qPCR (right) analysis of CD44ALDH1 cells with or without Spg-Let7.F,parentalCD44ALDH1 cells and cells with Spg-Let7 and Spg-Let7þshARID3B were transplanted subcutaneously in the neck region of immunocompromised mice (n ¼ 3; each mouse was injected by 106 cell). The tumor volumes of xenograft (mean tumor volume cm3 SEM) were measured by a caliper and monitored up to 6 weeks. , P < 0.05 versus control.

ARID3B (Fig. 4A and B). Luciferase reporter assays demonstrated studies demonstrated that the 30-UTR of HMGA2 contains seven that Let7 reduced the luciferase activity of reporter plasmids sequences complementary to the Let7 family of miRNAs (31), containing full-length ARID3B 30-UTR (Fig. 4C). However, when allowing Let7 to negatively regulate HMGA2 mRNA and protein the potential Let7-targeting site was deleted or mutated, Let7 no expression. In our OSCC model, we also found that Let7 sup- longer affected the luciferase activity (Fig. 4C). Consistently, the pressed HMGA2 expression through binding on its 30-UTR (Sup- protein and mRNA levels of ARID3B were decreased in the plementary Figs. S2F and S2G and S3). presence of Let7 (Fig. 4D) and increased in the absence of Let7 (Fig. 4E). These results identiﬁed a crucial Let7-binding site on the ARID3B and HMGA2 regulate the promoter activities of Oct4 30-UTR of ARID3B for Let7 to suppress its expression. To further and Sox2, respectively, through direct promoter binding link this regulatory mechanism to tumorigenesis of OSCC, we To investigate whether ARID3B directly regulates Oct4 expres- subcutaneously transplanted parental CD44 ALDH1 , CD44 sion, we searched for potential AT-rich regions in the proximal ALDH1 /Spg-Ctrl, CD44 ALDH1 /Spg-Let7, and CD44 promoter of Oct4 and found several potential binding sites of ALDH1 /Spg-Let7þshARID3B cells in the neck region of immu- ARID3B (Fig. 5A). We then constructed a series of Oct4 promoter- nocompromised mice and monitored the tumor growth up to driven luciferase reporter plasmids containing either full-length 6 weeks. Spg-Let7 enhanced the tumor growth, while shARID3B promoter, promoter with different length of deletions (D1-3), inhibited the Spg-Let7–induced tumor growth (Fig. 4F). Previous or promoter with mutations in the potential binding sites

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A B AATAAAAATAA TSS Putative Arid3B binding sites

TCAATAAA AAATAAA AATAAAAATAA TSS Full length –212 –202 –673 –666 –377 –371 –212 –202 TSS Arid3B binding site D1 pLV-Ctrl pLV-Arid3B –377 –371 –212 –202 TSS D2 -ARID3B -ARID3B None IgG α None IgG α –212 –202 TSS Full length D3 TCAATAAA AAATAAA AGCAAAAGCAA TSS D1 Mut –673 –666 –377 –371 –212 –202 D2

1007550250 D3 Relative changes of activity (%)

Mut

CD5′-AATAAAAATAA-3′ 5′-AGCAAAAGCAA-3′ TCAATAAA AAATAAA AATAAAAATAA Unlabeled probe −− Unlabeled MutProbe −− –673 –666 –377 –371 –212 –202 ARID3B + + + ARID3B + + + N.C. ARID3B RE1 ARID3B RE2 ARID3B RE3 − − Biotin-labeled probe + + + + Biotin-labeled MutProbe + + + + 10

8 ARID3B 6 ARID3B 4

2 Fold relative change 0

N.C. Free probe

ARID3B RE1 ARID3B RE2 ARID3B RE3 Free probe

Figure 5. ARID3B regulated the promoter activity of Oct4 through direct promoter binding. A, schematic presentation of the constructed full-length, deleted, and mutated Oct4 promoter-driven reporter plasmids. The full-length Oct4 promoter contains three AT-hook regions. The reporter plasmids were þ þ cotransfected with Let7 in CD44 ALDH1 OSCC cells and the luciferase activity was assessed and is presented in the graph. B, ChIP analysis of Arid3B at Oct4 þ þ promoter region. Empty vector or ARID3B-overexpressing plasmid was cotransfected with Oct4 promoter constructs in CD44 ALDH1 OSCC cells and subjected to a ChIP assay using anti-ARID3B antibody. IgG antibody was used as a background control. The Northern blot of the ChIP assay is presented in the ﬁgure. C, the qPCR analysis of ChIP assay with four different ampliﬁcation regions (NC and ARID3B RE1-3) is presented. Input, 2% of total lysate. D, EMSA and competition assays were performed using Arid3B, biotin-labeled, or unlabeled probes containing the Arid3B binding sequence of Oct4 promoter. Probes containing mutated Arid3B-binding site were used as negative control. Unlabeled probe were added at concentrations 10-fold (lane 3) or 50-fold (lane 4) greater than biotin-labeled probe. , P < 0.05 versus full length.

(Mut; Fig. 5A). Reporter assays showed that ARID3B induced the glioblastoma cells (26). This mechanism has not been shown in luciferase activity of full-length, D1, and D2 Oct4 promoter OSCC cells, however. Using the same approaches, we confirmed reporters, but not others (Fig. 5A). These data indicated that that HMGA2 bound to and increased the activity of Sox2 pro- ARID3B positively regulated the promoter activity of Oct4 and moter (Supplementary Fig. S4). Taken together, our results dem- this regulation depended on a specific sequence within the Oct4 onstrated a novel ARID3B-mediated Lin28B/Let7 effect on the promoter. A chromatin immunoprecipitation (ChIP) assay regulation of Oct4 expression through a direct binding between showed that Oct4 promoters were coprecipitated with ARID3B. ARID3B and Oct4 promoter. However, when the ARID3B-binding sequences were mutated or removed from Oct4 promoter, the coprecipitation was no longer The Lin28B–Let7 pathway regulates the stemness-driven observed (Fig. 5B). The association between ARID3B and Oct4 reprogramming in normal oral keratinocytes and OSCC cells promoter was further confirmed by a qPCR of the ChIP assay Reprogramming process has become a method to evaluate (Fig. 5C). Moreover, an electrophoretic mobility shift assay the strength of subject factors in regulating ESC-stemness and (EMSA) further supported the binding of ARID3B to the specific self-renewing property (31). Yu and colleagues (32) reported regions of Oct4 promoters (Fig. 5D). In addition, we previously that using a combination of Oct4, Sox2, Nanog, and Lin28 reported that HMGA2 regulates the expression of Sox2 through a (OSNL) is able to reprogram human somatic cells into iPSC. direct promoter binding, leading to enhanced CSC properites in Stemcellsandcancercellssharesimilarpropertiessuchasself-

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Lin28/Let-7 Reprogramming OSCC into Stem-like State

renewal and the expression of stemness signature (33). Aber- Stemgent), PD0325901 (0.5 mmol/L; Stemgent), and thiazo- rant reprogramming and epigenetic alteration deregulate stem- vivin (0.5 mmol/L; ref. 25). The iPSCs were later transferred ness properties and result in the induction of cancer stem–like to feeder-free/serum-free culture system (37). First, we executed properties and tumor development (34–36). Because Oct4 and a reprogramming protocol (23) on NHOK with OSN Sox2 mediate the ESC/stemness-acquisition, we then aimed (Oct4þSox2þNanog), OSNþLin28B, and OSNþSpg-Let7, to investigate the roles of Lin28B–Let7 signaling in cellular based on Yu and colleagues' protocol (32), and compared reprogramming in NHOK primary cells (21) and OSCC cells the reprogramming efﬁciency (Fig. 6A). As expected, the using a reprogramming protocol as described in the Sup- OSNþLin28B displayed the best reprogramming efﬁciency, plementary Data. In brief, iPSCs were reprogrammed from while OSN failed to reprogram NHOK without Lin28B. NHOK cells via the transduction of pMXs vectors encoding To our interest, Spg-Let7 had the same effect as Lin28B in the transcription factors Oct4, Sox2, Klf4, and c-Myc using a cooperating with OSN to reprogram NHOK. Co-overexpression virus infection system (23). On day 7 of after infection, target of Let7 in all the three reprogramming experiments dramati- cells were passaged onto mitotically inactivated MEF feeder cally reduced the iPSC colonies. These results suggested that layers, and cultured using human ESC medium. The iPSC suppression of Let7 is a crucial checkpoint for NHOK repro- colony formation was aided by adding SB431542 (2 mmol/L; gramming. We further examined the role of the Lin28B–Let7

A Oct4+Sox2+Nanog CD NHOK (Oct4+Sox2+Nanog+Lin28) pLV-Ctrl OSN+Lin28B OSN+Spg-Let7 Oct4+Sox2+KIF4+c-Myc NHOK.1 NHOK.1-iPSC NHOK.2 NHOK.2-iPSC ShLin28B ShCtrl ShLin28B + SpgLet-7

Lin28B Oct4

Oct4 Nanog

Sox2 – – NHOK CD44 /ALDH1 (+Lin28) Pt.1 Pt.1+Lin28 Pt.2 Pt.2+Lin28 3.5 Vector Ctrl KIF4 pLV-Let-7 3.0 c-Myc 2.5 Oct4 2.0 GAPDH 1.5 1.0 Nanog 0.5 0.0 Relative reprogramming efficiency Relative reprogramming OSN OSN+Lin28 OSN+Spg-Let7 F E NHOK (Oct4+Sox2+Nanog+Lin28) CD44+/ALDH1+ CD44–/ALDH1–

B 350 1.25 ShCtrl ShLin28B pLV-Ctrl pLV-Lin28B Oct4+Sox2+KIF4+c-Myc No. 1 No. 1 300 ShCtrl + ShLin28B + ShLin28 + No. 2 1.00 No. 2 SpgCtrl Spg-Ctrl Spg-Let7 250 0.75 U6 mRNA exp. 200 mRNA exp.

150 0.50 Let7 Lin28 100 0.25 50 100op Relative Relative 0 0.00 miR-98 NHOK NHOK-iPSC NHOK NHOK-iPSC i 1.25 g 90op CD44–/ALDH1– (+Lin28) f 1.00 80op 1.25 45 No. 1 d 40 No. 1 70op 0.75 No. 2 1.00 No. 2 35 c 30 0.50 0.75

mRNA exp. b efficiency 25 mRNA exp. 60op 20 0.25 0.50 Let7 Lin28 15

Relative reprogramming 0.00 10 0.25 a 50op ShCtrl + ShLin28B + ShLin28B + 5 Relative Relative 0 0.00 SpgCtrl Spg-Ctrl Spg-Let7 CD44-ALDH1CD44-ALDH1- CD44-ALDH1CD44-ALDH1- Oct4+Sox2+KIF4+c-Myc (+Lin28) (+Lin28)

Figure 6. The Lin28B–Let7 pathway mediated reprogramming efficiency of NHOK cells. A, NHOK cells were subjected to reprogramming procedure using Oct4þSox2þNanog (OSN), OSNþLin28B, or OSNþSpg-Let7 with or without exogenous Let7. The iPSC colonies were stained with alkaline phosphate and photographed (top). The quantification of iPSC colony number is presented at the bottom. B, a Oct4þSox2þKlf4þcMyc (OSKM) reprogramming protocol was performed in NHOK in the presence or absence of shLin28B and shLin28BþSpg-Let7. The iPSC colonies were stain and photographed (top). The number of colonies was calculated and is presented as relative value in the graph (bottom). C, the iPSC colonies from B were analyzed by Western blot analysis. D, bisulfite sequencing analysis of Oct4 and Nanog promoter regions in NHOK, NHOK-iPSC, CD44ALDH1/Ctrl, and CD44ALDH1/Lin28B cells. E, quantitative PCR analysis of Lin28B and Let7 levels in NHOK, NHOK-iPSC, CD44ALDH1/Ctrl, and CD44ALDH1/Lin28B cells. F, CD44þALDH1þ/ þ þ shCtrl, CD44 ALDH1 /shLin28B, CD44 ALDH1 /Ctrl, and CD44 ALDH1 /Lin28B cells were subjected to a SL-PCR analysis to assess the changes of Let7 family (including Let7a, Let7b, Let7c, Let7d, Let7f, Let7g, and Let7i) expression level. miR98, a miRNA with a similar sequence to Let7, was used as a control to show the distinguishability of the method.

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pathway in Yamanaka 4-gene (OSKM; Oct4þSox2þKlf4þ OSCC cells, and Oct4 and Sox2 are important down- cMyc) reprogramming protocol (38) and showed that knock- stream mediators of Lin28B/Let7/ARID3B/HMGA2 in regulat- down of Lin28B hampered the reprogramming efficiency as ing OSCC tumorigenesis. well as the protein expression levels of OSKM, while co-knock- In OSCC clinical specimens, IHC analysis further showed that down of Let7 partially recovered it (Fig. 6B and C). patient samples expressing high level of Lin28B also expressed Alteration of DNA methylation pattern of stemness genes is an ARID3B, HMGA2, Oct4, and Sox2 proteins (Fig. 7E) but low important epigenetic hallmark as well as a crucial barrier for cell level of Let7 (Fig. 7F). Analysis on a cohort of clinical specimens reprogramming (39). We compared the bisulfite sequencing revealed that more than 78% of Lin28Bhigh/Let7low patients results of Oct4 and Nanog promoters in NHOK and OSNL-driven expressed high level of ARID3B, HMGA2, Oct4, and Sox2; while HNOK-iPSCs, and showed that the Oct4 and Nanog promoters only less than 22% expressed low levels of these molecules were dramatically demethylated in NHOK-iPSC, compared with (Fig. 7G). Collectively, these data indicate a clinical correla- NHOK cells (Fig. 6D). Moreover, overexpression of Lin28B in tion between Lin28B-high, Let7-low, ARID3B-high, Oct4-high, CD44 ALDH1 OSCC cells also partially demethylated Oct4 HMGA2-high, and Sox2-high expression in OSCC patients, promoter but had little effect on Nanog promoter, compared supporting the clinical importance of Lin28B-Let7-ARID3B/ with nontransfected CD44 ALDH1 OSCC cells (Fig. 6D). This HMGA2-Oct4/Sox2 signaling in OSCC patients. may indicate an incomplete reprogramming in OSCC cells, con- tributing the switch from non-CSC to CSC state. Moreover, the Discussion results of qPCR analysis in Fig. 6E confirmed a pattern of high expression level of Lin28B and low expression level of Let7 in CSCs share numerous features with normal stem cells, the NHOK-iPSC and CD44 ALDH1 /Lin28B. In addition, Let7 including hallmark properties, such as self-renewal and undif- family members have recently been shown to regulate cancer ferentiated state, and CSCs were suggested to be arisen from progression and self-renewal in stem cells (8, 40, 41). To further non–stem-like cancer cells through a reprogramming mech- explore whether Lin28 could precisely regulate the endogenous anism very similar to the generation of iPSC (5). Both iPSC expression of Let7 family in OSCC, the size-coded ligation-medi- and CSC present unlimited proliferation and self-renewing ated PCR (SL-PCR), a method for detecting multiple miRNAs activities (43), and share similar characteristics of cell metab- þ þ simultaneously in total RNA (42), was used in CD44 ALDH1 olism. Moreover, many of the key transcription factors, such and CD44 ALDH1 OSCC cells. The SL-PCR data confirmed as Oct4 and Sox2, mediating the induction of iPSC and the the downregulation of endogenous Let7 family upon Lin28B maintenance of stemness phenotype in embryonic and soma- overexpressing in CD44 ALDH1 cells, while knockdown of tic stem cells are also overexpressed in CSC (4, 18, 44). þ þ Lin28B in CD44 ALDH1 cells elevated the endogenous Let7 Recently, Ohnishi and colleagues (5) demonstrated that pre- family (Fig. 6F). Taken together, these results strongly suggested mature termination of the reprogramming process leads to that Lin28B/Let7 signaling plays a crucial role in cell reprogram- cancer development in various tissues through altered epige- ming and regulates the acquisition of ESC-stemness properties netic regulation. We believe that the premature reprogram- both in NHOK and OSCC cells. ming is highly related to the teratoma formation of iPSC, as well as the development of CSC. In this study, we found a Clinical significance and potential application of the Lin28B- correlation of high Lin28B level and low Let7 level in CSC-like Let7-Arid3a/HMGA2-Oct4/Sox2 signaling in OSCC patients OSCC cells. Furthermore, we demonstrated that the Lin28B– To evaluate whether Lin28B reprograms the tumorigenesis Let7 pathway regulates cancer stemness in OSCC and plur- properties in OSCC, we preinjected GFP-labeled CD44 ipotency in NHOK through mediating Oct4 and Sox2 expres- ALDH1 /Lin28B and CD44 ALDH1 /Spg-Let7 cells trans- sion. The Lin28B/Let7-dependent regulation of Oct4 and Sox2 fected with shARID3B or shARID3B/shHMGA2 in immuno- provides further insights of the complex stemness regulatory compromised mice. Through monitoring GFP signal, we found networks. Further investigation may reveal its roles in regu- that overexpressed Lin28B or knockdown of Let7 increased the lating key stemness pathways during reprogramming, differ- tumor volume of CD44 ALDH1 cells, whereas co-knockdown entiation, and tumorigenesis. of ARID3B and HMGA2 decreased it (Fig. 7A and B). Moreover, We identified ARID3B as a novel downstream target of Let7. þ þ the tumor growth of highly tumorigenetic CD44 ALDH1 cells ARID3B is involved in survival of neural crest during embryo- was suppressed by shLin28B but rescued by co-overexpression genesis and is expressed in a portion of neuroblastoma (45–47); it of ARID3B and HMGA2 (Fig. 7C, top). The overexpression synergized with Mycn to control ESC proliferation (48). Recent ARID3B and HMGA2 plasmids used in this study does not studies indicated that ARID3B participates in tumor development contain 30-UTR. Therefore, the overexpressed HMGA2 and and is highly expressed in differentiated layers of squamous ARID3B would not be suppressed by endogenous or exogenous epithelium in malignant tissues (49). We revealed a direct binding þ þ 0 Let7. Overexpressing Let7 in CD44 ALDH1 cells was able to of Let7 in the 3 -UTR region of ARID3B mRNA, resulting in suppress their tumor growth, but with co-overexpression of suppressed ARID3B expression. ARID3B directly binds to Oct4 ARID3B and HMGA2, the Let7-suppressed tumor growth was promoter, confirmed by ChIP assay, and subsequently enhanced recovered (Fig. 7C, bottom). These tumor growth rates were in the mRNA and protein expression of Oct4. In response to Lin28B line with the expression levels of Oct4 and Sox2 in the tumor expression, suppression of Let7 allows ARID3B binding to a sections (Fig. 7D). Notably, our therapeutic experiments indi- specific sequence in Oct4 promoter and promote its transcription. 0 cated that delivery of synthetic hsa-Let7 may possess therapeutic Our data also confirmed that Let7 directly targets the 3 -UTR of potential in OSCC patients (Supplementary Fig. S5). These HMGA2, which upregulates Sox2 expression through promoter data showed that Lin28B, as well as its downstream effectors binding in response to Lin28B overexpression or Let7 suppres- ARID3B, were able to increase the tumorigenicity in non-CSC sion. The Let7-suppressed tumor-initiation and self-renewal

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− − − − CD44 /ALDH1 CD44 /ALDH1 ABEHLin28-high expression (Lin28hi) 1.0 ) 3 0.8 HE Lin28B pLV-Lin28B pLV-Lin28B pLV- +ShARID3B/ Lin28B 0.6 pLV-Ctrl HMGA2 +ShARID3B 0.4

0.2

Tumor volume (cm volume Tumor 0.0

HMGA2 Sox2 pLV-Lin28B Vector Ctrl pLV-Lin28B +ShARID3B pLV-Lin28B /HMGA2 +ShARID3B − − CD44 /ALDH1 1.0 ) 3 0.8 0.6 ARID3B Oct4 0.4

0.2

0.0 Tumor volume (cm volume Tumor

Vector Ctrl pLV-5pg-Let7 +ShARID35pLV-5pgLet7 20 /HMGA2 +ShARID3BpLV-5pg-Let7 F CDCD44+/ALDH1+ + + 15 CD44 /ALDH1 Ct) −Δ 1.6 ShCtrl Oct4 10 ) 1.25 Sox2 3 1.4 ShLin28B/HMGA2/ARID3B ShLin28B/HMGA2 1.00 5 1.2 of Let7 (2 ShLin28B 0.75 Relative expression Relative 1.0 0 0.50 high lo

0.8 in tumors Lin28B Lin28B 0.25 0.6 0.00 G Relative mRNA levels Relative 0.4 hi lo lo hi hi lo lo hi ShCtrl + -- - Lin28 Let7 Lin28 Let7 Lin28 Let7 Lin28 Let7 0.2 Sh-Lin288 - + - + Tumor volume (cm volume Tumor - 0.0 HMGA2 - + + --- Negative 123456 ARID3B + HMGA2 13% (6/46) 91% (40/44) Sox2 Negative 22% (10/46) 91% (40/44) Weeks CD44+/ALDH1+ 1.2 pLV-Ctrl Oct4 Positive 87% (40/46) 9% (4/44) Positive 78% (36/46) 9% (4/44) ) 1.25 3 pLV-Let7/HMGA2/ARID3B Sox2 1.0 pLV-Let7/HMGA2 1.00 P < 0.01 Fisher’s exact test P < 0.01 Fisher’s exact test pLV-Let7 0.8 0.75 Lin28hiLet7lo Lin28loLet7hi Lin28hiLet7lo Lin28loLet7hi 0.6 0.50 in tumors 0.4 0.25 0.00 ARID3B Negative 17% (8/46) 93% (41/44) Oct4 Negative 13% (6/46) 82% (36/44) 0.2 mRNA levels Relative pLV-Ctrl + --- Positive 83% (38/46) 7% (3/44) Positive 87% (40/46) 18% (8/44) Tumor volume (cm volume Tumor 0.0 pLV-Let7 --+ + HMGA2 --+ + 123456 --- P < 0.01 Fisher exact test P < 0.01 Fisher exact test Weeks ARID3B +

Figure 7. Clinical signiﬁcance of the Lin28B–Let7–ARID3B–HMGA2–Oct4–Sox2 pathway in OSCC. A and B, immunocompromised mice were transplanted in the neck subcutaneous region with 106 cells of GFP-labeled CD44 ALDH1 cells stably transfected with Lin28B, shARID3B, shHMGA2, or Spg-Let7 as indicated. The xenograft tumors were incubated for 6 weeks and the tumor size was visualized by GFP signal and quantiﬁed by a caliper. C and D, an in vivo tumor growth assay was performed with stable CD44þALDH1þ cell lines as indicated in immunocompromised mice (mean tumor volume cm3 SEM). The tumor growth was monitored up to 6 weeks, and the tumor sections were analyzed by qPCR for their Oct4 and Sox2 expression. E, patient specimens expressing high level of Lin28B were analyzed by IHC staining to assess the protein expression of ARID3B, HMGA2, Oct4, Sox2, and Lin28B. One representative patient specimen is presented. F, a qPCR analysis of Let7 mRNA levels in a cohort of Lin28Bhigh and Lin28Blow patient samples. G, IHC analysis of 46 Lin28BhighLet7low and 44 Lin28BlowLet7high OSCC patient samples for their expression of HMGA2, ARID3B, Oct4, and Sox2. The negative and positive expression of each of the four molecules were calculated and statistically correlated in the two groups of patient samples. H, schematic presentation of the proposed Lin28B–Let7–ARID3B–HMGA2–Oct4–Sox2 pathway in the current study.

capability was rescued by overexpression of ARID3B and HMGA2, cancer recurrence (39, 50). Premature reprogramming was which also rescued the suppressed Oct4 and Sox2, respectively. demonstrated to be leading to cancer development through We believe that in OSCC, Lin28B upregulates stemness transcrip- altering epigenetic signatures (5). In our data, the Lin28B–Let7 tion factors, such as Oct4 and Sox2, as well as self-renewal and pathway not only mediates cancer stemness, but also regulates tumor-initiation ability, through increased ARID3B and HMGA2. cellular reprogramming. The Lin28B–Let7 pathway interferes Clinical specimens revealed a significant correlation between with reprogramming efficiency in NHOK-derived iPSCs Lin28B-high, Let7-low, ARID3B-high, Oct4-high, HMGA2-high, through regulating Oct4 and Sox2 expression. Modulating the and Sox2-high in OSCC patients. Most importantly, therapeutic Lin28B/Let7 signaling altered the gene expression signature and delivery of Let-7-mimics in orthotropic-transplanted NOD/SCID pluripotency of iPSC, methylation patterns of Oct4 and Nanog, mice reduced the tumor growth (Supplementary Fig. S5). Collec- and shifted the gene expression pattern between the dynamic tively, these results demonstrate a Lin28B/Let7-driven ARID3B/ change of stem-like and non–stem-like states in both NHOK Oct4 signaling pathway that activates the stemness-derived tumor and OSCC cells. Through demethylating Oct4 and Nanog initiation and malignance during oral cancer formation. promoters, Lin28B-mediated pathway enhanced reprogram- The pathologic reprogramming process in cancer has been ming efficiency of NHOK. It has to be noted that the effect of shown to be critical in modulating the CSC induction and Lin28B on Nanog promoter demethylation seemed very little in

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Chien et al.

OSCC cells, implying a partial or premature reprogramming. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, The role of Lin28B/Let7 signaling in stemness pathway and computational analysis): C.-S. Chien, M.-L. Wang, W.-H. Liu, Y.-T. Lan, pluripotency regulation during cell reprogramming echoes with P.-I. Huang, S.-H. Chiou Writing, review, and/or revision of the manuscript: C.-S. Chien, M.-L. Wang, its role in cancer stemness, and it would be interesting to W.-H. Liu, Y.-T. Lan, S.-H. Chiou investigate the demethylation mechanism of stemness gene Administrative, technical, or material support (i.e., reporting or organizing promoters in the Lin28B/Let7-modulated reprogramming data, constructing databases): Y.-L. Chang, Y.-T. Lan, P.-I. Huang, Y.-W. Chen, process. S.-H. Chiou Conclusively, we reported a Lin28B–Let7 pathway in OSCC; Study supervision: Y.-L. Chang, S.-H. Chiou through ARID3B/HMGA2–mediated Oct4/Sox2 signaling, this pathway enhanced the ESC-stemness properties, tumor initiation, Grant Support and malignance of OSCC. This study would greatly contribute to a This study was assisted, in part, by the Division of Experimental Surgery of the Department of Surgery and the Animal Center of Taipei Veterans deeper understanding of cancer reprogramming and ESC-stem- General Hospital. This study was funded by the NSC & MOST (102-2314-B- ness acquisition in OSCC, and promote the development of 075-015-MY3, 103-2627-M-010-004, and 103-2321-B-010-025), Taipei effective therapies for OSCC eradication. Veterans General Hospital (Stem Cell Project E99-101), The Department of Health Cancer Center Research of Excellence (MOHW104-TDU-B-211- 124-001), National Health Research Institutes (NHRI-EX102-10258SI), Disclosure of Potential Conﬂicts of Interest NRPB Human iPSC Alliance-Core Service (MOST104-2325-B-001-010), and No potential conﬂicts of interest were disclosed. The Genomic Center Project and Cancer Center Project of National Yang- Ming University (Ministry of Education, Aim for the Top University Plan), Taiwan. Authors' Contributions The costs of publication of this article were defrayed in part by the Conception and design: C.-S. Chien, M.-L. Wang, Y.-T. Lan, Y.-Y. Lee, W.-L. Lo, payment of page charges. This article must therefore be hereby marked S.-H. Chiou advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Development of methodology: C.-S. Chien, W.-H. Liu, C.-C. Yu, Y.-T. Lan this fact. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C.-S. Chien, P.-Y. Chu, W.-H. Liu, Y.-T. Lan, Y.-Y. Lee, Received July 30, 2014; revised January 15, 2015; accepted March 7, 2015; W.-L. Lo published OnlineFirst April 9, 2015.

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www.aacrjournals.org Cancer Res; 75(12) June 15, 2015 2565

Lin28B/Let-7 Regulates Expression of Oct4 and Sox2 and Reprograms Oral Squamous Cell Carcinoma Cells to a Stem-like State

Chian-Shiu Chien, Mong-Lien Wang, Pen-Yuan Chu, et al.

Cancer Res 2015;75:2553-2565. Published OnlineFirst April 9, 2015.

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