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 À À 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. Ó2015 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 prop- erty 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 Ó2015 American Association for Cancer Research. the self-renewal of ESCs, and promotes the malignant www.aacrjournals.org 2553 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst April 9, 2015; DOI: 10.1158/0008-5472.CAN-14-2215 Chien et al. 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 Â 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 Â 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 Â 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