Department of Dentistry, MacKay Memorial Hospital, Taipei, Taiwan › content › canres › early › ... · are described in Supplementary methods. 100 ug/ml of 4NQO was added in

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MicroRNA-211 enhances the oncogenicity of carcinogen-induced oral carcinoma by repressing TCF12 and increasing antioxidant activity

Yi-Fen Chen1, Cheng-Chieh Yang1-3, Shou-Yen Kao2,3, Chung-Ji Liu2,4, Shu-Chun Lin1-3＊, Kuo-Wei Chang1-3*

1Institute of Oral Biology and 2Department of Dentistry,

National Yang-Ming University, Taipei, Taiwan. 3Department of Stomatology, Taipei Veterans General Hospital, Taipei, Taiwan

4Department of Dentistry, MacKay Memorial Hospital, Taipei, Taiwan

*Corresponding authors: Shu-Chun Lin, PhD Institute of Oral Biology School of Dentistry National Yang-Ming University No. 155, Li-Nong St., Section 2 Taipei, Taiwan 112 Fax: +8862-28264053 E-mail: [email protected]

Kuo-Wei Chang, DDS, PhD Department of Dentistry School of Dentistry National Yang-Ming University No. 155, Li-Nong St., Section 2 Taipei, Taiwan 112 Fax: +8862-28264053 E-mail: [email protected]

Running title: miR-211-TCF12-FAM213A activation in OSCC Conflict of interest: The authors declare no conflict of interest. Grant support: Shu-Chun Lin received grant MOST102-2628-B-010-006-MY3 from Ministry of Science and Technology. Kuo-Wei Chang received grant V103C-070 from Taipei Veterans General Hospital and grant 104AC-P504 from Aim for the Top University Plan from Department of Education. Shou-Yen Kao, Kuo-Wei Chang, and Cheng-Chieh Yang received grant for Health and Welfare Surcharge of tobacco products number MOHW104-TD-B-111-02 from Ministry of Health and Welfare for Excellence for Cancer Research. Keywords: FAM213A, HNSCC, microRNA, miR-211, Oral Cancer, TCF12

The authors assure that we will pay the color charge fees for Figures 1 and 7.

on July 4, 2020. © 2016 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 24, 2016; DOI: 10.1158/0008-5472.CAN-15-1664

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Abstract

miR-211 expression in human oral squamous cell carcinoma (OSCC) has been

implicated in poor patient survival. To investigate the oncogenic roles of miR-211, we

generated K14-EGFP-miR-211 transgenic mice tagged with green fluorescence

protein. Induction of oral carcinogenesis in transgenic mice using 4-nitroquinoline

1-oxide (4NQO) resulted in more extensive and severe tongue tumorigenesis

compared with control animals. We found that 4NQO and arecoline upregulated

miR-211 expression in OSCC cells. In silico and experimental evidence further

revealed that miR-211 directly targeted transcription factor 12 (TCF12), which

mediated suppressor activities in OSCC cells and was drastically down-regulated in

tumor tissues. We used GeneChip analysis and bioinformatic algorithms to identify

transcriptional targets of TCF12 and confirmed through reporter and ChIP assays that

FAM213A, a peroxiredoxin-like anti-oxidative protein, was repressed

transcriptionally by TCF12. FAM213A silencing in OSCC cells diminished oncogenic

activity, reduced the ALDH1-positive cell population and increased reactive oxygen

species. TCF12 and FAM213A expression was correlated inversely in head and neck

carcinoma samples according to The Cancer Genome Atlas. OSCC patients bearing

tumors with high FAM213A expression tended to have worse survival. Furthermore,

4NQO treatment down-regulated TCF12 and up-regulated FAM213A by modulating

miR-211 both in vitro and in vivo. Overall, our findings develop a mouse model that

recapitulates the molecular and histopathological alterations of human OSCC

pathogenesis and highlight a new microRNA-mediated oncogenic mechanism.




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Introduction

Exposure to carcinogenic substances or viruses are the major etiological factors

of head and neck squamous cell carcinoma (HNSCC) including oral squamous cell

carcinoma OSCC (1-3). Many factor such as arecoline, are oxidative inducers (3). The

five-year survival of OSCC remained low and this malignancy tended to relapse after

treatment (4). Therefore, it is important to specify the molecular dis-regulation in the

regulatory axis contributive to OSCC pathogenesis to develop therapeutic modalities

(5). MicroRNAs (miRNAs) are small, non-coding RNAs of 19-25 nucleotides, which

regulate physiological process or pathogenesis by targeting the mRNA to cause

transcriptional repression or mRNA degradation (6,7). A number of disruptions in

miRNA-target gene regulatory axes in OSCC have been discovered (8-13).

miR-211 promoted OSCC oncogenicity and it served as an indicator for poor

OSCC survival (14). We also showed that miR-211 targets TGFßRII, which is able to

up-regulate c-myc expression in HNSCC (15). miR-211 also functions as an

oncogenic miRNA in colorectal cancer (CRC) by targeting CHD5 (16). In addition,

miR-211 promotes cell growth by targeting the p53 induced loc285194 LncRNA in

CRC (17). A recent study has also specified that miR-211 prevents the ER induction

by transcriptional repression of CHOP to modulate the survival of cells (18). Despite

that miR-211 is a tumor suppressor in melanoma and some other cancers (19-21), the




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function of miR-211 as an oncogenic molecule has become more evident in a fraction

of malignancies (15-18).

Transcription factor 12 (TCF12) (22), belongs to class I helix-loop-helix (HLH)

protein family known as E protein, which can bind to DNA E-box site (23,24). TCF12

was shown to regulate the differentiation of lymphocytes or the development of neural

or mesenchymal tissues (25-28). Recurrent mutations in TCF12 gene or the

translocation fusion of a fragment of TCF12 with other molecules contribute to

craniosynostosis or mesenchymal malignancies (29,30). In CRC, TCF12 expression

correlated with metastasis by repression of E-cadherin (22). TCF12 has been reported

targeted by miR-154 and miR-211 in melanoma cells and other types of normal cells

(20,31,32). Nevertheless, the roles of TCF12 in OSCC and other cancers are still

obscure.

Reactive oxygen species (ROS) are intracellular chemical species that contain

oxygen. Accumulated ROS causes oxidative stress and may induce cytotoxicity in

cells (33), antioxidants are considered to be suppressors against cancers, as cancer

cells seem to possess a higher tolerance of ROS than normal cells. However, recent

evidence showed that the up-regulation of antioxidant protein Nrf2 might promote

survival and resistance to therapies in cancer cells (34). A subpopulation of HNSCC

cells carrying low ROS may exhibit more stemness and chemoresistance properties




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(35).

Peroxiredoxin (PRX) and thioredoxin (TRX) are two related families of

antioxidant proteins. PRXs uptake H2O2 to become oxidized form. The oxidized

PRXs then reduced by TRXs (36,37). Family with sequence similarity 213, member A

(FAM213A) was discovered as one of the members in the PRX-like subfamily.

Moreover, it also possesses an essential domain for TRXs activation (36). FAM213A

was originally identified during fetal liver development and was activated in M-CSF

stimulated monocytes (38). It was later found to protect cells from oxidative stress

and modulates osteoclast differentiation (39). A recent study indicated that FAM213A

could be one of the candidate antioxidants beneficial for high-altitude adaptation in

Andean people (40).

4-nitroquinoline 1-oxide (4NQO) is a water-soluble carcinogen, which breaks

DNA and induces ROS (41,42). The murine 4NQO tongue carcinogenesis has become

a powerful model to address oral carcinogenesis (13,15). To address the oncogenic

roles of miR-211 in the animal model, we generated miR-211 Tg mouse lines driven

by the K14 promoter, which were tagged with enhanced green fluorescence protein

(EGFP). We identified the enhancement of OSCC progression by 4NQO induction in

this mouse model. Furthermore, we identified transcription factor TCF12 as a new

target of miR-211 in OSCC cells. The enhanced FAM213A expression mediated by




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the repression of TCF12 through miR-211 expression reinforces OSCC oncogenesis

and protects cells from the oxidative damages.

Materials and Methods

Cells

OSCC cell lines SAS, OECM1, HSC3, FaDu, OC4, and SCC25; 293T cells and

phoenix package cells; and six primary OSCC cells isolated from different tumors

were used. Cell lines were achieved from ATCC or JCRB cell banks or derived

according to previous protocols during 2012 - 2014 (11,13,15). All cell lines were

authenticated by short tandem repeat analysis. The cultivation condition are described

in Supplementary Table S1. SAS-miR-211, OECM1-miR-211, and control cell

expressing GFP were established previously (14). The treatment conditions of

miR-211 mimic/inhibitor/control (Applied Biosystems, Foster City, CA) were 60 or 30

nM for 48 or 72 h treatment. The dose of siTCF12 (Dharmacon, Lafayette, CO) and

siFAM213A (Santa Cruz Biotech, Santa Cruz, CA) oligonucleotides were 60 nM for

30 or 48 h (Supplementary Table S2). All chemicals were purchased from

Sigma-Aldrich (St Louise, MO). siRNAs and TaqMan® assay probes are described in

Supplementary Table S2 and S3.

Phenotypic analysis




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Phenotypes including proliferation, migration, wound healing, invasion,

anchorage-independent growth (AIG), and ALDEFLUOR assay followed protocols

previously published (11,13,43,44).

Generation of K14-GFP-miR-211 transgenic mice and tumor induction

The murine pri-miR-211 sequence and EGFP were cloned to establish Tg mouse

lines in C57BL/6 (Supplementary Table S4) (13). For genotyping, genomic DNA

isolated from mouse tail tip were used for PCR and Southern blot analysis. The RNA

isolated from mouse ear was used for gene transcription analysis (15). Other details

are described in Supplementary methods. 100 ug/ml of 4NQO was added in drinking

water of 6-8 week-old mice for 16 weeks. Mice were sacrificed at the time point when

body weight loss >1/3, or at the defined time points (13,41).

Orthotopic and subcutaneous xenograft

Three x 105 SAS cells were injected into the central portion of the tongue of

BALB/c athymic mice (National Laboratory Animal Center, Taipei, Taiwan). The

mice were sacrificed at the third week after inoculation. The primary tongue tumors

and neck region were photographed under the Illumatool Bright Light System

(LT-9500; TLS, Sarasota, FL) to visualize the positive tumor and nodes with green

fluorescence. Tongue and neck tissues were subjected to histopathological evaluation.

Five x105 SAS cells were injected into the flank of BALB/c athymic mice. The




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tumors were measured every week and the mice were sacrificed at the sixth week

after inoculation. The tumor volumes were calculated by the formula = 0.5xaxb2 using

parameters measured by micro-scale under light microscopy or gauge (8). a, the

longest diameter; b, the shortest diameter. All animal studies were done in accordance

with the guideline of National Yang-Ming University Institutional Animal Care and

Use Committee (IACUC).

Plasmids and establishment of stable cell subclones

The TCF12 coding sequence (CDS), TCF12 CDS plus 3’UTR (WT), and TCF12

CDS plus mutated 3’UTR were cloned into the pBabe-puro vector to produce

retroviral constructs (Supplementary Table S4). The TCF12 expression cells were

designated CDS, WT, and MUT, together with VA (vector alone) control. Short

hairpin shTCF12 constructs (Supplementary Table S5) packed in lentiviruses were

purchased from the RNA interference consortium (Academia Sinica, Taipei, Taiwan).

Cell exhibiting the knockdown of TCF12 were designated b4 and b5 together with

shLuc (control).

OSCC tissue samples and tissue microarray (TMA)

Primary OSCC tumors together with paired non-cancerous matched tissues

(NCMT) (Supplementary Table S6) were collected for qRT-PCR and Western blot

analysis. The OSCC TMAs encompassing paired NCMT/OSCC tissue cores or OSCC




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tumor cores only (Supplementary Table S7) were fabricated to carry out

immunohistochemistry (IHC) and in situ hybridization (ISH) analysis (45). Detailed

methods were described in Supplementary methods and Table S8. This study was

approved by institute review board (IRB) with approval No. 2013-11-011B and

12MMHIS177. Written informed consents were obtained from participants.

GeneChip analysis

GeneChip® Human Genome U133 Plus 2.0 arrays (Affymetrix, Santa Clara, CA)

were used. Qualified RNA was submitted to National Yang-Ming University VGH

Genome Research Center (VYMGC) for GeneChip analysis. The accession number is

GSE70186.

Transcription factor binding sites analysis

Jaspar (http://jaspar.genereg.net), an open-access database of the transcription

factors binding preferences in multiple species (46), was used to predict potential

transcription factor binding sites.

Statistical analysis

The data were shown as Mean ± S.E. t-test, Mann-Whitney test, X2 test, two-way

ANOVA test, linear regression analysis, and Kaplan-Meier survival analysis were

used to compare the differences among variants. ns, not significant; *, p<0.05; **,

p<0.01; ***, p<0.001.




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Results

miR-211 expression is associated with more advanced oncogenesis and metastasis

We generated the mmu-miR-211-based Tg mouse model. The schematic diagram

(Fig. 1A, a) illustrates the K14-EGFP-miR-211 transgene construct, which results in

constitutive expression of EGFP and miR-211 driven by K14 promoter in squamous

cells. The characterization of these Tg mouse lines is shown in Supplementary Fig. S1.

Increased the thickness of cell layers, as well as increased expression of Ki67 and

Bcl-xL are seen in squamous epithelium of Tg mice (Supplementary Fig. S2 and Fig.

S3). We induced tumorigenesis by adding 4NQO in the drinking water (Fig. 1A, b).

The treatment successfully induced tumors on the tongue surface, in the esophagus

and occasionally on the palate or buccal mucosa. The tumors with intensified green

fluorescence on dorsal tongue were identified easily in Tg mice. Moreover, the tumors

in un-opened esophagi were discernable rather readily due to their intensive

fluorescence (Fig. 1A, c). The incidence of tongue tumor number and tumor size were

significantly higher in Tg mice than Wt mice (Fig. 1A, d). Histopathological

evaluation showed epithelial hyperplasia or dysplasia in normal looking tongue

mucosa (Fig. 1B, a, Upper Lt and Middle panels). Tissue sections of exophytic lesions

showed pathogenesis varied from squamous cell papilloma (SCP), moderate to severe

epithelial dysplasia (Dys), squamous cell carcinoma with submucosal invasion




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(SCC-S) to SCC with muscle invasion (SCC-M) (Fig. 1B, a). The quantitation

revealed increased severity of squamous pathogenesis following the increase of

transgene dosage (Fig. 1B, b). Tg mice exhibited shorter survival than Wt mice for 1.7

weeks (Fig. 1B, c). The results suggest that the K14-EGFP-miR-211 Tg mice have

higher susceptibility to 4NQO for oral tumor induction than Wt mice. The esophageal

tumor induction is shown in Fig. 1A, c and Supplementary Fig. S4.

Orthotopic xenograft model of SAS-miR-211 was further adopted to address

primary tumorigenesis and locoregional metastasis in nude mice. The xenografic

tumors in tongue and neck metastatic lesions were demonstrated by fluorescence

image and histopathological analysis (Fig. 1C, a, b; Supplementary Fig. S5). miR-211

expression was associated with higher tumor growth and higher percentage of nodal

metastasis (Fig. 1C, c, Lt and Middle). In the primary tumors subset exhibiting a size

<10 mm3, the locoreginal metastasis of SAS-miR-211 xenografts were also more

potent than controls (Fig. 1C, c, Rt). miR-211 expression rendered higher neck

metastasis of OSCC.

miR-211 targeted TCF12 in OSCC cells

To examine if oncogenic factors can stimulate the miR-211 expression, we

treated OSCC cells with 4NQO or arecoline. 4NQO treatment for 48 h resulted in

miR-211 up-regulation in OSCC cells (Fig. 2A, a, Lt). The induction was mediated by




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the increase of pri-miR-211 transcript (Supplementary Fig. S6). With the treatment of

various doses of arecoline for 24 h, miR-211 expression was also up-regulated in SAS

cell (Fig. 2A, a, Rt). mRNA expression and reporter activity of potential miR-211

targets CDH4, HAS2, KLLN and UBA2 predicted by in silico modules and TCF4,

TCF12 and TGFẞRII known targets were assayed (Fig. 2A, b, c). The mRNA

expression and reporter activity of HAS2, KLLN, TCF4, TCF12 and TGFẞRII were

decreased in OSCC cells with exogenous miR-211 expression. However, 4NQO

treatment only consistently down-regulated TCF12 in OSCC cells (Fig. 2A, d).

Down-regulation of TCF12 mRNA and protein expression were found in SAS cells

with exogenous miR-211 expression (Fig. 2A, e). FaDu, HSC3, and OECM1 cells had

lower TCF12 expression than SAS cell, but higher miR-211 expression than SAS (Fig.

2B, a). Supplementary Table S9 illustrates the complementarity between the TCF12

3’UTR sequence and the miR-211. We generated a wild-type TCF12 3’UTR reporter

(wt) and a mutant reporter (mut). Reporter assays in OSCC cells indicated that

miR-211 repressed the reporter activity of TCF12 by directly targeting the wild-type

3’UTR sequence, and that the mutation relieved the repression (Fig. 2B, b).

Down-regulation of TCF12 expression in the squamous epithelium of Tg mice

relative to the Wt mice was noted (Fig. 2B, c). In oral epithelium from Wt to Tg/+,

then to Tg/Tg mice, progressive increase of miR-211 expression was noted. However,




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the nuclear TCF12 expression gradually decreased (Fig. 2B, d; Supplementary Fig.

S7). In OECM1, the CDS and the MUT had higher TCF12 expression than the WT.

The WT had slightly higher TCF12 expression than the control. Exogenous miR-211

expression had more profound repression of TCF12 in the WT than the MUT (Fig. 2C,

a, b). HSC3 had the highest miR-211 expression and lowest TCF12 expression. The

CDS had much higher TCF12 expression than WT. (Fig. 2C, a). The results

implicated that the 3’UTR sequence can create an opportunity for miR-211 repression,

which substantiated the targeting of miR-211 on TCF12. The efficacy of shTCF12

constructs was validated in SAS (Fig. 2C, c). Stable shTCF12 cells b4 and b5 were

established in SAS and OECM1 (Fig. 2C, c, d). To further confirm the repression of

TCF12 by miR-211, we treated SAS cells with miR-211 inhibitor. The up-regulation of

TCF12 protein mediated by miR-211 inhibition was rescued by the knockdown of

TCF12 (Fig 2D, a, Upper). The down-regulation of TCF12 expression in

SAS-miR-211 cell was also rescued by miR-211 inhibitor (Fig. 2D, a, Lower). The

analysis of nuclear extract and the cytosolic fraction indicated that the vast majority of

cellular TCF12 localized in nuclei, and its levels corresponded to the fluctuation of

miR-211 levels being modulated by miR-211 mimic or inhibitor (Fig. 2D, b;

Supplementary Fig. S8). Therefore, the nuclear TCF12 immunoreactivity in tissue is

likely a true signal. Since the percentages of TCF12 in nucleus did not change




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conspicuously following miR-211 modulation, miR-211 expression was unable to

regulate the translocation of TCF12.

TCF12 mediated the tumor suppressor effects on OSCC cells

In OECM1 cells, neoplastic activities including proliferation, migration, invasion

and AIG were decreased in TCF12 WT cell, and such activities further reduced in

MUT cell (Fig. 3A, a). However, except for AIG, other oncogenic activities in CDS

were similar to WT. It appeared that OECM1 CDS was rather resistant to TCF12

driven oncogenic suppression (Supplementary Fig. S9). In HSC3 cells, the neoplastic

activities of CDS were lower than those in WT (Fig. 3A, b). The increased migration

in OECM1-miR-211 was repressed by TCF12 expression (Fig. 3A, c). OECM1 with

the knockdown of TCF12 exhibited increased neoplastic activities (Fig. 3B, a). Apart

from the in vitro oncogenic modulation, SAS with the knockdown of TCF12

increased subcutaneous tumorigenicity in vivo (Fig. 3B, b). The clues indicate that

TCF12 mediates suppressor activity against OSCC in general. The reduced neoplastic

activities resulted from the miR-211 inhibition were rescued by the knockdown of

TCF12 (Fig. 3C). Thus, miR-211 induces OSCC oncogenicity by targeting the TCF12

tumor suppressor.

Down-regulation of TCF12 mRNA expression was found in 78% (39/50) of

human OSCC tumors as compared to their paired NCMTs (Fig. 3D, a). TCF12 protein




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expression was unequivocally lower in OSCC tumors relative to the NCMTs (Fig. 3D,

b). It appeared that miR-211 expression in the NCMTs was higher than the

corresponding OSCC tumors, and there was no correlation between miR-211

expression and TCF12 mRNA expression in OSCC tumors (Supplementary Fig. S10).

To clarify this ambiguity, miR-211 staining and nuclear TCF12 staining were

performed in human OSCC. It indicated a reverse association between miR-211

expression and TCF12 expression (Fig. 3D, c; Supplementary Fig. S11). Besides, the

increase of miR-211 staining in tongue epithelium from Wt to heterozygous, and to

homozygous Tg mice was noted. 4NQO treatment enhanced such increase. On the

contrary, there was progressive decrease of nuclear TCF12 staining in tongue

epithelium from Wt to Tg mice. 4NQO treatment enhanced such decrease during the

murine multistep carcinogenesis (Supplementary Fig. S7 and Fig. S12). The relatively

high miR-211 expression in NCMTs revealed by qRT-PCR could be resulted from the

intensive submucosal miR-211 expression (Supplementary Fig. S11A).

Identification of FAM213A as a downstream effector of TCF12

To investigate the regulation of TCF12 on downstream genes, we performed

GeneChip analysis using OECM1 knockdown cells and HSC3 expression cells. A

total of 31 and 3 presumed oncogenic and suppressor gene spots were identified (Fig.

4A, a; Supplementary Fig. S13). Gene annotation and network analysis are shown in




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Supplementary Fig. S14. We selected 7 genes, NNMT, TNFFS10, ABCA, CLCA,

FAM213A, GDF15, and TRIM29 (Tripartite motif-containing protein 29), which either

have been shown in GeneChip for the presence of more than two spots or having the

Jaspar score of >50 for qRT-PCR analysis (Fig. 4A, b; Supplementary Fig. S15;

Supplementary Table S10). Changes in FAM213A and TRIM29 mRNA expressions

across three different cells were consistent (Supplementary Fig. S16). Analyses

applied on different OSCC cells showed that relative to controls, FAM213A and

TRIM29 were down-regulated in cells having exogenous TCF12 expression; while

they were up-regulated when TCF12 expression was knocked down (Fig. 4B, a). To

investigate if miR-211 regulates FAM213A and TRIM29, SAS cells were treated with

miR-211 mimic or inhibitor. The repression of TCF12 induced by miR-211 mimic

drastically up-regulated FAM213A, but it only slightly up-regulated TRIM29. The

up-regulation of TCF12 induced by miR-211 inhibition resulted in the

down-regulation of FAM213A and TRIM29 (Fig. 4B, b). The regulation of

miR-211-TCF12 axis on FAM213A was more eminent than TRIM29. In a panel of

OSCC cells, including 6 cell lines and 5 primary tumor cell cultures, miR-211

expression is positively correlated with FAM213A mRNA expression (Fig. 4C, a).

Down-regulation of TCF12 and up-regulation of FAM213A were noted in the stripped

tongue and skin epithelium of Tg mice in relation to Wt mice (Fig. 4C, b). Gene




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expression data from the NCI-60 database and The Cancer Genome Atlas (TCGA)

HNSCC database identified a reverse correlation between TCF12 and FAM213A in

cancer cell lines and HNSCCs (Supplementary Fig. S17). OSCC cells treated with

4NQO displayed miR-211 up-regulation (Fig. 2A, a, Lt), accompanied with the

down-regulation of TCF12 in mRNA (Fig. 4C, c) and protein level (Fig. 4D, a). This

TCF12 down-regulation was miR-211 associated (Fig. 4D, a). In addition, 4NQO

induced FAM213A up-regulation in OSCC cells was related to TCF12

down-regulation but was irrelevant with TCF4 or TGFẞRII (Fig. 2A, d; Fig. 4C, c

and Fig. 4D, b). The findings substantiate the existence of miR-211-TCF12-FAM213A

regulatory axis in OSCC, which is 4NQO inducible.

TCF12 down-regulates FAM213A oncogene through promoter repression

Combined analysis from the Jaspar database with manual precision mapping of

E-box elements defined 13 E-boxes on sense or antisense strand in the -1000-TSS

segment of human FAM213A gene. The region between -608 to -792 seemed to be a

hotspot as it contained 7 E-boxes (Fig. 5A, a). To specify that TSS~-1000 segment

could possess FAM213A promoter activity was analyzed. It showed an increased

luciferase activity in the reporter harboring FAM213A -1000-TSS segment with the

knockdown of TCF12 in SAS and HSC3 cells (Fig. 5A, b, Lt). Oppositely, OECM1

with TCF12 expression had lower reporter activity (Fig. 5A, b, Rt). To clarify if




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TCF12 directly binds to the hotspot region in the promoter, ChIP assay was carried

out to amplify the sequences being precipitated by anti-TCF12 antibody. Comparing

to the control, the use of anti-TCF12 antibody yielded amplicons in a dose-dependent

manner in HSC3 cell (Fig. 5A, c, Lt). Moreover, OECM1 expressing TCF12 also had

higher binding of TCF12 to this hotspot region relative to controls (Fig. 5A, c, Rt).

The results suggest that TCF12 is able to repress the promoter activity of FAM213A

in OSCC cells through the binding to this region containing an E-box cluster.

Knockdown of FAM213A was carried out in OSCC cells (Fig. 5B, a). FAM213A

knockdown significantly repressed the oncogenic phenotypes including proliferation,

migration, invasion and AIG in SAS and OECM1 (Fig. 5B, b). The effects on

migration, invasion, and AIG were more profound than proliferation. In OECM1 cell,

the migration being induced by the knockdown of TCF12 was repressed when

FAM213A was knocked down (Fig. 5B, c). Furthermore, the migration and invasion

induced by miR-211 were repressed when FAM213A was knocked down (Fig. 5C).

With the knockdown of FAM213A, the ALDH1-postive OSCC cell population

decreased by about 50% (Fig. 5D). Strong FAM213A staining was noted in human

OSCC tissues (Supplementary Fig. S18A). The progressive increase of FAM213A

expression was also noted during the 4NQO-induced multistep carcinogenesis

(Supplementary Fig. S18B). Thus, FAM213A, a downstream gene transcriptionally




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repressed by TCF12, plays an oncogenic role in OSCC.

FAM213A expression protects cells from oxidative stress

To stratify that the intracellular ROS was regulated by

miR-211-TCF12-FAM213A axis in OSCC cells, cells were challenged with H2O2 to

evoke ROS. The assays showed that exogenous miR-211 expression and the

knockdown of TCF12 decreased the ROS, whereas the knockdown of FAM213A

increased the ROS in OSCC cells (Fig. 6A). The effects of miR-211 in reducing ROS

were limited. To elucidate the impact of TCF12-FAM213A on the cell migration

being modulated by ROS, wound-healing assays were performed. The induction of

ROS drastically impeded the migration of SAS cells, while the knockdown of TCF12

attenuated such impedance (Fig. 6B, Lt). The induction of ROS slightly inhibited the

migration of OECM1 cells. Knockdown of TCF12 reverted such inhibition, and the

reversion could be further repressed by the knockdown of FAM213A (Fig. 6B, Rt).

Collectively, the results substantiate a role of FAM213A in abrogating

ROS-associated deleterious effects on cell migration. The formation of 8-OHdG in

nucleic acid and the genesis of carbonyl group in proteins are markers of oxidative

stress. The knockdown of FAM213A or the 4NQO treatment increased carbonyl

proteins (Fig. 6C). By staining the nuclear 8-OHdG and the carbonyl proteins, we also

detected more profound oxidative stress in tongue epithelium of Tg mice than Wt




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mice (Fig. 6D; Supplementary Fig. S19). Oxidative stress was particularly high in the

tumor tissues of Tg mice. Although there was high FAM213A expression in tumors of

Tg mice (Supplementary Fig. S18B), its scavenger efficiency was not sufficient for

ROS attenuation in epithelial tissue. It is likely that FAM213A contributes to

oncogenesis via other activity in addition to ROS scavenger.

Association between FAM213A expression and poor patient survival of OSCC

The nearly absent miR-211 staining was noted in the human NCMT tissues in

TMA (Fig. 7A, Upper; Supplementary Fig. S11A). There was stronger miR-211

staining in corresponding paired OSCC tissues. Remarkable miR-211 expression was

noted in stroma subjacent to the human oral epithelium. There was a complete

absence or relatively fainter FAM213A staining in NCMT tissues. However, the

cytosolic FAM213A staining in paired and unpaired tumor tissues were much stronger

(Fig. 7A, Lower; Supplementary Fig. S18A). Quantitation of the pixel readings

showed a significant increase of both miR-211 and FAM213A expression from NCMT

to OSCC (Fig. 7B, a, b), which was highly correlated (Fig. 7B, c). Although the

FAM213A expression in stage I - III tumors and stage IV tumors were not much

different in view of the pixel scoring (Fig. 7B, d), OSCC having strong FAM213A

expression exhibited a trend of worse overall and disease-free survival (Fig. 7B, e;

Supplementary Fig. S20A). In the OSCC at stage I - III, association between




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FAM213A expression and worse survival was more evident (Fig. 7B, f;

Supplementary Fig. S20B).

Representative analyses of miR-211 and FAM213A staining in tissues

harvested from Wt mice at different time points during the multistep carcinogenesis

were illustrated (Fig. 7C, a, b; Supplementary Fig. S18B). The progressive increase of

miR-211 and FAM213A expression followed the increased severity of epithelial

pathogenesis (Fig. 7C, c, d). The increase of miR-211 and FAM213A expression

occurred prior to neoplastic formation. In agreement with this tendency, nuclear

TCF12 expression decreased during the multistep carcinogenesis (Supplementary Fig.

S12). The findings confirm that 4NQO may modulate miR-211-TCF12-FAM213A

axis and contributes to the progression of OSCC. The schema in Fig. 7D depicts our

thought on how miR-211 targets TCF12, which then trans-inactivates FAM213A for

oral carcinogenesis.

Discussion

miR-211 is associated with the pathogenesis of several malignancies including

OSCC (14-17,19-21). Studies have elucidated that miR-211 is versatile in targeting

multiple genes in different kinds of cells (10,15-21,31,47). In addition to a miR-211

target being found in melanoma (20,31). TCF12 is also a miR-211 target in OSCC.




22

Although the oncogenic activity of HAS2 and KLLN appears irrelevant to

4NQO-miR-211 regulation, their pathogenic roles acting as new miR-211 targets need

further specification. The repression of TCF12 on the transcription of FAM213A may

account for a novel molecular mechanism underlying the OSCC oncogenesis as

induced by miR-211. We established K14-EGFP-miR-211 Tg mouse models. These Tg

mice have increased thickness, increased pro-survival protein Bcl-xL expression, and

endogenous oxidative stress in squamous epithelium, and a higher 4NQO associated

tumor induction. It is worth noting that the miR-211 dosage is also correlated with the

tumor burden and aggressiveness. These findings substantiate the contribution of

miR-211 for promoting squamous carcinogenesis. Due to the presence of GFP tag, this

design of genetic engineering has facilitated the rapid screening of infant mice

carrying transgene. With the assistance of fluorescence, some insidious tumors on the

tongue surface or embedded in the un-opened esophagus, not readily detectable with

visible light, were more clearly defined. Since mice with advanced tumors and poor

health are sacrificed before endpoint, this might underlie the limited tumorigenic

enrichment in Tg mice. The assessments of locoregional metastasis are also

confounded accordingly. We have demonstrated that miR-211 expression promotes

primary OSCC tumor growth and neck nodal metastasis in orthotopic nude mice

model (8).




23

This study identified that 4NQO can up-regulate miR-211 in oral keratinocytes

both in vivo and in vitro. Therefore, the enrichment of miR-211 expression induced by

4NQO enhances the severity of tumorigenesis in transgenic mice, could be a plausible

mechanism. The role of miR-211 in the human carcinogenesis process of esophagus,

skin and cervix is unclear. This animal model would be useful for assessing the

chemical- or viral-associated tumorigenesis in squamous epithelium other than oral

tongue. Due to the rather strong miR-211 expression in human stromal cells and the

versatility of miR-211 in targeting genes, the roles that miR-211 plays in stromal

pathogenesis require elucidation.

Our functional assays clarified that miR-211 targets TCF12 both in vitro and in

Tg mouse model. TCF12 functions to suppress OSCC oncogenicity. In addition, most

miR-211-associated tumor phenotypes are reversed by TCF12. As TCF12 is a negative

regulator, the exogenous TCF12 expression in OECM1 cell is limited, and the

resistance to TCF12 mediated suppression may emerge. TCF12 was conspicuously

down-regulated in the vast majority of OSCC tumors. As TCF12 has many spliced

variants and it is prone to get mutation or translocation in diseases (29,30), to further

define its functional implications and to characterize the genomic abnormalities in a

wide variety of malignancies is required. Antibodies more specifically detect nuclear

TCF12 activity are required to facilitate tissue studies. Our approaches pinpointed




24

FAM213A and TRIM29 as potential downstream effectors of TCF12. A fraction of

potential TCF12 targets may have been masked in the initial screening according to

our criteria. TRIM29 seems to be a key regulator of epithelial-mesenchymal transition

(EMT) and tumor invasion, but its roles among neoplasms are controversial. It

up-regulates CD44 and induces EMT in K-ras induced pancreatic carcinoma (48). On

the contrary, it also suppresses TWIST1 and inhibits EMT in breast carcinoma (49).

As miR-211 expression up-regulates TRIM29 rather limitedly, unclear factors may

exist to confound this regulation in OSCC. Although no effort has been made to

further delineate its interplay with TCF12, to resolve oncogenic stimuli that regulate

TCF12-TRIM29 would be important for understanding tumor invasion and EMT.

FAM213A attenuates ROS associated signals, which then elicits the

differentiation of bone marrow monocytes (39). Our approaches further unravel that

TCF12 down-regulates FAM213A through transcriptional repression (22), and

FAM213A depletion decreases oncogenicity. In addition to the fact that endogenous

miR-211 and FAM213A are rather synchronized, 4NQO stimulation also up-regulates

these oncogenic events concordantly. These clues signify a new

miR-211-TCF12-FAM231A regulatory axis in OSCC oncogenesis upon carcinogenic

stimulation. Despite that FAM213A has weak impact on proliferation relative to other

neoplastic activities, FAM213A expression increases from normal mucosa to




25

neoplasm in both human OSCC and murine carcinogenesis process, FAM213A is

contributive to the early establishment of OSCC. Our previous study identified the

prognostic values of miR-211 expression in OSCC (14), this study suggests the

potential of FAM213A expression in determining the survival of patients carrying

stage I-III tumors.

miR-211 was reported to reduce ER stress in CRC (18). miR-204, another

member of the miR-211 family, increases the sensitivity to oxidative stress in neuron

cells (47). This study specifies that miR-211 up-regulates FAM213A, which then

reduces ROS in OSCC cells. We also show that FAM213A may protect cancer cells

from the oxidative damages. Although the findings of increased oxidative stress in the

oral epithelium of Tg mice is to our surprise, the results may implicate a potential

linkage between miR-211 expression and the higher cellular injury for tumor

susceptibility. Since the antioxidative activity of miR-211 seems negligible, it is

unable to attenuate the oxidative stress. The up-regulation of proteins associated with

proliferation or survival in epithelium may be responded to the cell injury in

epithelium. FAM213A expression is highly associated with invasion and the increased

ALDH1-positive cell population. miR-211 is found to facilitate the neck metastasis of

OSCC xenografts in this study. As oxidative stress could hinder the distal metastasis

of melanoma cells (50), whether the ROS attenuation or the stemness induction




26

mediated by FAM213A can profit the metastatic dissemination need elucidation.

Since the vast majority of OSCCs have strong FAM231A expression, the therapeutic

efficacy of OSCC could be improved by incorporating anti-FAM213A strategy into

conventional chemotherapy.

In this study, we specify that miR-211 is an oncogenic regulator of OSCC by

targeting the TCF12 tumor suppressor, and TCF12 transcriptionally represses

FAM213A oncogenic molecule. 4NQO-miR-211-TCF12-FAM213A tends to be a

novel regulatory cascade for oral carcinogenesis. Whereas the activities of FAM213A

in enhancing specific oncogenic signals remained to be resolved, its dual properties

and high expression in OSCC may implicate targeting values (8,39). The

K14-EGFP-miR-211 Tg mice might be a suitable model to develop regimen for OSCC

intervention.

Acknowledgements

We thank Professor Tin-Fen Tsai and Ms. Courtney Anne Curtis for their

assistances.

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Figure legends

Fig 1. Induction of mouse tongue tumorigenesis and metastasis. A. (a) Transgenic

construct. (b) 4NQO treatment. Arrowheads, sampling time. Arrows, 4NQO treatment.

(c) Gross and fluorescence images of tongue and esophagus. Rt, dissected esophagus.

Red arrows, fluorescent lesions. Yellow arrow, lesions only illuminated by

fluorescence but not by gross inspection. (d) Quantitation of the lesion. Un-paired

t-test. B. (a) Histopathological sections of tongue from 4NQO treated mice. (x100).

Indent, deepest part of the lesion. (b) Quantitation of the severity of epithelial

pathology in 17, 47 and 33 exophytic lesions achieved from Wt, Tg/+, and Tg/Tg

mice. X2 test. (c) Kaplan-Meier survival analysis. C. (a, b) Fluorescence images and

histopathological sections of orthotopic xenografic tongue tumors and the associated

neck nodal metastasis. (c) Primary tumors, nodal metastasis, and nodal metastasis in

the smaller tumor subset. Mann-Whitney test.

Fig 2. miR-211 targets TCF12 in OSCC cells. A. (a) Lt, 4NQO treatment; Rt,

arecoline treatment in SAS. (b) mRNA expression and (c) reporter assays of potential

targets of miR-211. (d) mRNA expression of HAS2, KLLN, TCF4, TCF12, and

TGFßRII in 4NQO treated cells. (e) TCF12 mRNA and protein expression in

SAS-miR-211 and SAS cell with miR-211 mimic treatment. un-paired t-test. B. (a) Lt,




32

Endogenous TCF12 protein expression. Rt, Correlation between miR-211 and TCF12

protein expression in OSCC cell lines. (b) TCF12 wt and mut reporters in OSCC cells

with increased miR-211 expression. (c) The TCF12 protein expression in the stripped

squamous epithelium of Tg and Wt mice. (d) ISH and IHC. miR-211 staining and

nuclear TCF12 immunoreactivity in mouse tongue tissues. Numbers, pixel scores; %,

nuclear immunoreactivity. (x200). C. TCF12 protein expression. (a) CDS and WT of

OECM1 and HSC3. (b) WT and MUT of OECM1 with miR-211 mimic treatment or

not. (c, d) SAS and OECM1 shTCF12 cells. D. TCF12 protein expression. (a) SAS

treated with miR-211 inhibitor or the knockdown of TCF12. (b) Subcellular fractions.

%, nuclear or cytosolic percentage of TCF12.

Fig 3. TCF12 mediates suppressor activity in OSCC. A – C. Phenotypic analysis. A.

(a, b) OECM1 and HSC3 with TCF12 expression. (c) Migration. OECM1-miR-211

with TCF12 CDS expression. B. TCF12 knockdown cells. (a) OECM1. (b) SAS, Lt,

AIG; Rt, subcutaneous tumorigenesis. C. Cells treated with miR-211 inhibitor and/or

knockdown of TCF12. inh, inhibitor. Un-paired t-test or two-way ANOVA test. D.

Analysis of human OSCC sample pairs. (a) Before-after plot of TCF12 mRNA

expression. (b) Lt, Western blot. LN, metastatic neck node. Rt, Quantitation of 13

sample pairs. Paired t-test. (c) Linear regression analysis.




33

Fig 4. Identification of FAM213A as a downstream gene of TCF12. A. (a) Heat

map, 34 gene spots retrieved from GeneChip. A line separates the putative oncogene

group (Lt) from the putative suppressor group (Rt). (b) Venn diagram comparison.

White, TCF12 knockdown; Gray, TCF12 expression. The selected genes are subjected

to qRT-PCR analysis. B. FAM213A and TRIM29 protein expression (a) TCF12

expression or knockdown OSCC cells. (b) SAS cells treated with different dosages of

miR-211 mimic or inhibitor. C. (a) miR-211 expression and FAM213A mRNA

expression in OSCC cell lines and primary cultures. Linear regression analysis. (b)

TCF12 and FAM213A protein expression in squamous epithelium of mice. (c) TCF12

and FAM213A mRNA expression in 4NQO treated cells. Un-paired t-test. D. (a) Lt,

4NQO down-regulates TCF12 protein expression in OSCC cells. Rt, The

down-regulation is enhanced by miR-211 mimic and reverted by miR-211 inhibitor. (b)

4NQO treatment and the expression of TCF12, TCF4 and TGFẞRII in OSCC cells.

Fig 5. TCF12 down-regulates FAM213A through promoter inactivation. A. (a)

Predicted E-boxes in -1000-TSS of FAM213A gene. Arrows, primers for PCR

amplification. TSS, transcriptional start site. (b) Reporter assay in OSCC cells

transfected with siTCF12 or TCF12 CDS. (c) ChIP assay. Upper, Gel image. Lower,

qPCR analysis. Lt, HSC3 cells; Rt, OECM1TCF12 expression cell. B. (a, b)




34

Knockdown of FAM213A protein expression and phenotypic assays in OSCC cells. (c)

Rescue between TCF12 and FAM213A on the migration in SAS. C. Rescue between

miR-211 and FAM213A on the migration (Lt) and invasion (Rt) in SAS-miR-211. D.

ALDEFLUOR assay in FAM213A knockdown cells. Un-paired t-test or two-way

ANOVA test.

Fig 6. FAM213A expression protects cells from oxidative stress. A. OSCC cells

treated with miR-211 mimic, siTCF12 or siFAM213A are then treated with 1 mM

H2O2, and ROS levels are measured. B. Wound healing assay. Confluent OSCC cells

with the knockdown of TCF12 and/or FAM213A expression are treated with various

doses of H2O2 for 2 h and then wounded. The wound closure ratios represent the

migration capability. C. Western blot analysis detects the protein oxidation. D. IHC.

Lt, Nuclear 8-OHdG; Rt, Immunoreactivity of carbonyl proteins in mouse tongue

tissues (Upper panels), normal looking mucosas (Middle panels), and exophytic

lesions (Lower panels) in 4NQO treated mice. Indent, background immunoreactivity

(without adding DNPH). Un-paired t-test.

Fig. 7. FAM213A immunohistochemistry. A. miR-211 staining (Upper) and

FAM213A immunoreactivity (Lower) in human OSCC/NCMT tissue pairs. Arrows,




35

miR-211 staining in the stroma of NCMT. Indents, ISH staining using scramble probe.

(x200). B. (a, b) Before-after plot of miR-211 and FAM213A pixel scores in paired

samples. Paired t-test. (c) Correlation between miR-211 and FAM213A expression in

human OSCC. Linear regression analysis. (d) FAM213A expression in OSCC tumors

at different clinical stages. Classification of weak, moderate, and strong FAM213A

expression based upon the OSCC/NCMT ratio <1.1, 1.1-1.5 and >1.5. (e, f) Overall

survival of patients according to FAM213A expression. (e) All OSCC patients. (f)

Stage I - III patients. C. (a) miR-211 staining and (b) FAM213A immunoreactivity in

4NQO induced multistep carcinogenesis process. (x100). (Numbers), treatment weeks;

Numbers in A and C, pixel scores. (c, d) Quantitation of miR-211 and FAM213A

expression in different steps of tongue carcinogenesis. SCP, squamous cell papilloma;

Dys, moderate or severe dysplasia; SCC-S, SCC with submucosal invasion; SCC-M,

SCC with muscle invasion. Un-paired t-test. D. Summary of the

miR-211-TCF12-FAM213A regulatory axis in OSCC.

























Published OnlineFirst May 24, 2016.Cancer Res Yi-Fen Chen, Cheng-Chieh Yang, Shou-Yen Kao, et al. increasing antioxidant activitycarcinogen-induced oral carcinoma by repressing TCF12 and MicroRNA-211 enhances the oncogenicity of

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Department of Dentistry, MacKay Memorial Hospital, Taipei, Taiwan › content › canres › early › ... · are described in Supplementary methods. 100 ug/ml of 4NQO was added in