TRIM29 regulates the p63-mediated pathway in cervical cancer cells

BBAMCR-17596; No. of pages: 10; 4C: 4, 7 Biochimica et Biophysica Acta xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamcr

TRIM29 regulates the p63-mediated pathway in cervical cancer cells

2Q2

Yasushi Masuda, Hidehisa Takahashi, Shigetsugu Hatakeyama ⁎

3

Department of Biochemistry, Hokkaido University Graduate School of Medicine, Kita 15, Nishi 7, Kita-ku, Sapporo, Hokkaido 060-8638, Japan

4

a r t i c l e

5 6 7 8 9

Article history: Received 27 November 2014 Received in revised form 14 May 2015 Accepted 26 May 2015 Available online xxxx

10 11 12 24 13 14 15

Keywords: TRIM29 Cell adhesion Cervical cancer p63 Integrin

O

a b s t r a c t

R O

i n f o

37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

E T

C

E

R

35 36

R

33 34

Invasion and migration are significant hallmarks of metastatic cancer cells. Epithelial to mesenchymal transition (EMT) allows cancer cells to acquire metastatic ability [1]. During the EMT process, interaction of integrins with the extracellular matrix (ECM) exerts a prominent role by regulating the function of matrix metalloproteinases (MMPs) [1]. The degradation of ECM components by MMPs contributes to cancer metastasis by changing the tumor microenvironment [1]. These biological processes are orchestrated by a network of transcription factors including SNAIL1/2, TWIST1/2 and ZEB1/2 [1]. In addition to these transcription factors, one of the members of the p53 family, p63, is involved in cancer metastasis by regulating the expression of integrins [2,3]. Uterine cervical cancer is one of the common health problems for women worldwide [4]. Human papillomavirus (HPV) is a significant risk factor associated with invasive cervical cancer [5]. Among the various HPV types, HPV-18 is strongly related to adenocarcinoma of the uterine cervix, whereas HPV-16 is detected more frequently in squamous cell carcinoma than in adenocarcinoma [6,7]. HPV infection enhances the invasion activity of cervical cancer cells by upregulation of MMPs [8]. In cervical cancer cells, epidermal growth factor promotes the transition of epithelial cells to mesenchymal cells, being dependent on SNAIL1 and α5β1 integrin [9]. Interaction of α5β1 integrin with fibronectin contributes to the migration with up-regulation of MMP-9 in cervical cancer cells [10]. Although EMT-related genes contribute to metastasis of cervical cancer, how cervical cancer cells gain invasive ability remains to elucidate.

N C O

31 32

1. Introduction

U

30

16 17 18 19 20 21 22 23

D

P

Cell invasion and adhesion play an important role in cancer metastasis and are orchestrated by a complicated network of transcription factors including p63. Here, we show that a member of the tripartite motif protein family, TRIM29, is required for regulation of the p63-mediated pathway in cervical cancer cells. TRIM29 knockdown alters the adhesion and invasion activities of cervical cancer cells. TRIM29 knockdown and overexpression cause a significant decrease and increase of TAp63α expression, respectively. TRIM29 knockdown alters the expression pattern of integrins and increases ZEB1 expression. TRIM29 is required for suppression of an increase in the adhesion activity of cells by TAp63α. These findings suggest that TRIM29 regulates the p63-mediated pathway and the behavior of cervical cancer cells. © 2015 Published by Elsevier B.V.

28 26 25 27 29

F

1Q1

⁎ Corresponding author. E-mail address: [email protected] (S. Hatakeyama).

TRIM29 has been identified as an ataxia telangiectasia group Dcomplementing gene in a functional complementation study with an AT cell line by transfection with a human cosmid library [11]. TRIM29 is a member of the tripartite motif (TRIM) protein family and is specifically expressed in squamous cells [12,13]. There is increasing evidence that TRIM29 enhances or suppresses cell invasion depending on cell type. In breast cancer cells, TRIM29 suppresses invasiveness by downregulating the expression of TWIST1 [14]. TRIM29 promotes cell invasion by regulating MMP-9 in lung cancer [15]. TRIM29 also regulates the invasion of pancreatic cancer cells by inducing EMT [16]. Furthermore, it has been reported that overexpression of TRIM29 is associated with malignancy of various cancers including bladder, colorectal, gastric lung and pancreatic cancers [12]. To date, it has been controversial whether TRIM29 functions as an oncogene or tumor suppressor. Although expression of TRIM29 is closely related to the characteristics and malignancy of cancer, the relationship between TRIM29 and cervical cancers remains incompletely understood. In this study, we identified TRIM29 as a regulator of the p63mediated pathway in cervical cancer cells. TRIM29 was required for the suppression of ZEB1 and integrin β1 (ITGB1). Depletion of TRIM29 suppressed an elevation of the adhesion activity of cells by TAp63α. We propose that TRIM29 regulates the p63-mediated pathway and the behavior of cervical cancer cells.

55

2. Materials and methods

78

2.1. Plasmid

79

56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77

Full-length human TRIM29 (NM_012101.3) was amplified using 80 total RNA from HeLa cells. TAp63α-FLAG was a gift from David 81 Sidransky (Addgene plasmid # 27008). 82

http://dx.doi.org/10.1016/j.bbamcr.2015.05.035 0167-4889/© 2015 Published by Elsevier B.V.

Please cite this article as: Y. Masuda, et al., TRIM29 regulates the p63-mediated pathway in cervical cancer cells, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbamcr.2015.05.035

Y. Masuda et al. / Biochimica et Biophysica Acta xxx (2015) xxx–xxx

89 90

97 98

HeLa, HeLa S3 and SiHa cell lines were obtained from American Type Culture Collection (Manassas, VA), which authenticates human cell lines in their collection, and were cultured in DMEM (Sigma-Aldrich Corp., St Louis, MO) supplemented with 10% fetal bovine serum (FBS) under an atmosphere of 5% CO2 at 37 °C. Unless otherwise indicated, cells were cultured in a 100-mm plastic dish (Corning 430167; Corning Co., NY, USA). For large-scale cultures, parental and FLAG-tagged TRIM29-expressing HeLa S3 cells were cultured in Joklik's medium (Sigma) supplemented with 5% calf serum (Invitrogen) using spinner culture.

99

2.4. Antibodies

100 101

108 109

Mouse anti-FLAG M2 was purchased from Sigma (Sigma). Mouse anti-TRIM29 (A-5: sc-166718), rabbit anti-p63 (H-137: sc-8343), mouse anti-p53 (DO-1: sc-126) rabbit anti-Pol II (N-20: sc-899) and mouse anti-actin (C-2: sc-8432) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA.). Rabbit anti-Integrin β4 (4707) was purchased from Cell Signaling Technology (Beverly, MA). Rabbit antiIntegrin β1 (ab52971) was purchased from Abcam (Cambridge, UK). Mouse anti-GAPDH (AM4300) was purchased from Ambion (Austin, TX). Rabbit anti-MED12 was purchased from Bethyl Laboratories (Bethyl Laboratories, Montbomery, TX).

110

2.5. siRNA transfections

111

116

HeLa S3, HeLa and SiHa cells in 10-cm dishes (~2 × 106 cells/dish) were transfected with 25 nM siRNA ON-TARGETplus human TRIM29 siRNA SMART pool (Dharmacon, Pittsburgh, PA) or 25 nM siGENOME NON-TARGETING siRNA Pool #2 (Dharmacon) using Lipofectamine RNAiMAX (Invitrogen). After 48-h culture, cells were subjected to each analysis.

117

2.6. Quantitative real-time PCR

118

Total RNA was extracted using ISOGEN II (Nippon Gene, Tokyo, Japan). cDNA was prepared by using oligonucleotide (dT) and a ReverTra-Plus kit (TOYOBO, Japan) for quantitative real-time PCR (qRT-PCR). The threshold cycle (Ct) values were determined by qRTPCR using Express SYBRGreenER qPCR Supermix Universal (Invitrogen) on an ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA). ΔCt was obtained by subtracting the Ct value of the GAPDH gene from the Ct value of the respective gene. The relative expression level of mRNA was determined by 2−ΔCt. All PCR primers were as follows: TRIM29, 5′-AAAGGCTATCCCTCCCTCAT-3′ (forward) and 5′-TAGAATGGCCGGTAGTGAGA-3′ (reverse); p63, 5′-GCAGCTTT GCAAACC CATTA-3′ (forward) and 5′-GCTCGGAAGTCCTAGGTTTATT-3′ (reverse); TAp63, 5′-TGTATCCGCATGCAGGACT-3′ (forward) and 5′CTGTGTTATAGGGACTGGTGGAC-3′ (reverse); ΔNp63, 5′-GAAAACAA TGCCCAGACTCAA-3′ (forward) and 5′-TGCGCGTGGTCTGTGTTA-3′ (reverse); p63α, 5′-ATTGATGCTGTGCGATTCACC-3′ (forward) and 5′TGTTGCTTATTGCGGCGAG-3′ (reverse); p63β, 5′-AACGCCCTCACTCCTA CAAC-3′ (forward) and 5′-GACTTGCCAGATCCTGACAATG-3′ (reverse); p63γ, 5′-GCAGCACCAGCACTTACTTCA-3′ (forward) and 5′-TGTTTTGG AGTTTCTCTCCGG-3′ (reverse); SNAIL2, 5′-TTTCTGGGCTGGCCAAACAT AAGC-3′ (forward) and 5′-ACACAAGGTAATGTGTGGGTCCGA-3′ (reverse); ZEB1, 5′-CAGGAAAGGAAGGGCAAGAA-3′ (forward) and 5′-

114 115 Q3

119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139

C

E

112 113

R

106 107

R

104 105

O

102 103

C

95 96

N

93 94

U

91 92

2.7. Cell adhesion assay

173

Cell adhesion assay to fibronectin, collagen I, collagen IV, laminin I and vimentin was performed using a CytoSelect 48-well cell adhesion assay extracellular matrix (ECM) array (Cell Biolabs Inc., San Diego, CA) according to the manufacturer's instructions. Cells were incubated on the ECM array for 1 h under an atmosphere of 5% CO2 at 37 °C in a serum-free medium. Each well was gently washed to remove nonadherent cells. Adherent cells were stained by Cell Stain Solution. The stains were extracted by Extract Solution and quantified by measuring absorbance at 595 nm.

174 175

2.8. Cell invasion assay

183

Cell invasion assay was performed using a CytoSelect 24-well cell invasion assay kit (Cell Biolabs Inc.) according to the manufacturer's instructions. Cells were incubated on the upper chamber on a polycarbonate membrane coated with basement membrane matrix in a serum-free medium. Cells were incubated for 1 h under an atmosphere of 5% CO2 at 37 °C. Non-invasive cells were gently removed from the interior of the chamber. Cells that had invaded to the bottom of the membrane were stained by Cell Stain Solution. The stains were extracted by Extract Solution and quantified by measuring absorbance at 595 nm.

184

2.9. Immunostaining

193

Cells were cultured on a cover glass in DMEM supplemented with 10% FBS under an atmosphere of 5% CO2 at 37 °C. The cells were fixed by 4% paraformaldehyde in PBS for 15 min at room temperature, washed with PBS three times, and then incubated with blocking buffer (5% normal goat serum and 0.1% Triton X-100 in PBS) for 1 h. After blocking, the cells were incubated with primary antibodies in blocking

194

F

2.3. Cell culture

O

88

R O

86 87

cDNAs encoding full-length TRIM29 and TAp63α were introduced by retroviral transduction into a HeLa S3 cell line stably expressing the mouse ectopic retrovirus receptor (mCAT-1) [17]. Retroviruses were produced with Plat-E packaging cells [18].

140 141

P

84 85

TCTGCATCTGACTCGCATTC-3′ (reverse); p53, 5′-TTTCGACATAGTGTGG TGGTG-3′ (forward) and 5′-GCCCATGCAGGAACTGTTA-3′ (reverse); ITGB1, 5′-CCTGAAAGTCCCAAGTGTCAT-3′ (forward) and 5′-ACCAACAC GCCCTTCATT-3′ (reverse); ITGB4, 5′-GAGCTGCATCGGCTCAA-3′ (forward) and 5′-AACACGTAGGAGTGGTTGG-3′ (reverse); ITGA3, 5′-AAAC TGTGGAGGATGTAGGAAG-3′ (forward) and 5′-CACTCCAGACCTAGGA CCA-3′ (reverse); ITGA5, 5′-GGGACCTCAGATCCTGAAATG-3′ (forward) and 5′-CTGCAGACTTTGGCTCTCTT-3′ (reverse); ITGA6, 5′-AGACTCTT AACTGTAGCGTGAAC-3′ (forward) and 5′-ATAAGAGACGCCTTGCTGTC3′ (reverse); FN1, 5′-AGGAATATCTCGGTGCCATTT-3′ (forward) and 5′CTTCGGGACTGGGTTCAC-3′ (reverse); VIM, 5′-ATCCAAGTTTGCTGAC CTCTC-3′ (forward) and 5′-CTCAGTGGACTCCTGCTTTG-3′ (reverse); SOX2, 5′-TACAGCATGATGCAGGACCA-3′ (forward) and 5′-TACTGCAG GGCGCTCAC-3′ (reverse); RB1, 5′-GGATGGAGTATTGGGAGGTTAT-3′ (forward) and 5′-AGTGAACGACATCTCATCTAGG-3′ (reverse); FOXM1, 5′-AAGATGAGTTCTGATGGACTGG-3′ (forward) and 5′-AAGGCTCCTC AACCTTAACC-3′ (reverse); E2F1, 5′-GGAGAAGTCACGCTATGAGAC-3′ (forward) and 5′-GTCGACGACACCGTCAG-3′ (reverse); E2F2, 5′-GTCC CTGAGTTCCCAACC-3′ (forward) and 5′-GAAGTGTCATACCGAGTCTTCT3′ (reverse); E2F3, 5′-AGGCTGGAGCTAGGAGAAA-3′ (forward) and 5′CGTAGTGCAGCTCTTCCTTT-3′ (reverse); E2F4, 5′-CCGGGAGATTGCTG ACAAA-3′ (forward) and 5′-CCTTGTGCTGGTCTAGTTCTT-3′ (reverse); E2F5, 5′-ACCAATGTCTTAGAGGGAATTGA-3′ (forward) and 5′-TTTAGT ATTACAGCCAGCACCT-3′ (reverse); E2F6, 5′-TCCAAGAACCATATTAGA TGGATAGG-3′ (forward) and 5′-ATTAACTCATCCAAAGCATCTTCC-3′ (reverse); HOXB5, 5′-TCCGCAAATATTCCCCTGGA-3′ (forward) and 5′CGGGTCAGGTAGCGGTTGAA-3′ (reverse); HOXB7, 5′-GACTTGGCGGCG GAGAGTAA-3′ (forward) and 5′-CAGGGTCTGGTAGCGGGTGT-3′ (reverse); HOXB9, 5′-CCACTGGCTGCTGGACTGTT-3′ (forward) and 5′TATGACTGGGCCAGGTTCTTTG-3′ (reverse); BRCA1, 5′-GCATGCTGAA ACTTCTCAACC-3′ (forward) and 5′-CGTACTTTCTTGTAGGCTCCTT-3′ (reverse); GAPDH, 5′-GTCAACGGATTTGGTCGTATTG-3′ (forward) and 5′-TGTAGTTGAGGTCAATGAAGGG-3′ (reverse).

D

2.2. Generation of stable cell lines

T

83

E

2

Please cite this article as: Y. Masuda, et al., TRIM29 regulates the p63-mediated pathway in cervical cancer cells, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbamcr.2015.05.035

142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172

176 177 178 179 180 181 182

185 186 187 188 189 190 191 192

195 196 197 198 199

Y. Masuda et al. / Biochimica et Biophysica Acta xxx (2015) xxx–xxx

207

212 213

Cells were transfected with siControl or siTRIM29 and incubated on fibronectin-coated coverslips in serum-free medium. After 48 h, cells were fixed and stained with anti-actin and anti-ITGB1 antibodies. DNA was counterstained with DAPI (0.4 μg/ml). Cell adhesion area was calculated as the ratio of ITGB1-stained area to DAPI-stained area. ITGB1- and DAPI-stained areas were quantified using NIH ImageJ software (n = 20).

214

2.11. FLAG immunoprecipitation

215 216

Nuclear extracts of HeLa S3 cells expressing FLAG-tagged TRIM29 were prepared according to Dignam's method [19]. Anti-FLAG affinity purifications were performed as described previously [20].

A

To reveal the function of TRIM29 in cervical cancer cells, we knocked down TRIM29 in HeLa S3 (HPV-18-positive), HeLa (HPV-18-positive) and SiHa (HPV-16-positive) cells by siRNA transfection and then performed an in vitro cell adhesion assay using plates coated with fibronectin, fibrinogen, collagen I, collagen IV or laminin I. TRIM29 knockdown in HeLa S3 cells increased the adhesion activity of cells to fibronectin (4.4-fold), fibrinogen (2.6-fold), collagen IV (1.7-fold) and collagen I (1.5-fold) (Fig. 1A), and TRIM29 knockdown in HeLa cells increased in adhesion activity to fibronectin (2.2-fold), collagen IV (2.7-fold) and collagen I (3.8-fold) (Fig. 1B). TRIM29 depletion in SiHa cells did not have a significant effect on adhesion activity (Fig. 1C). It has been reported that TRIM29 regulates invasion of lung and breast cancer cells [14–16]. To examine whether TRIM29 regulates cell invasion, we performed an in vitro cell invasion assay using polycarbonate membranes coated with basement membrane matrix. TRIM29 knockdown reduced the invasion activity of HeLa S3 and SiHa cells (Fig. 1D). In addition, we confirmed a reciprocal relationship between cell morphology and TRIM29 knockdown in each cell line. Knockdown of TRIM29 caused a significant change in morphology of HeLa S3 cells but not morphology

220 221

B

C

siCTRL

siCTRL 0.25

0.15

* *

*

0.15 0.1

R

N C O

siTRIM29 0.25

0

R

0

D

*

0.05

E

0.05

HeLa S3

*

C

0.1

0.2

E

0.2

T

Absorbance at 595 nm

*

siCTRL

siTRIM29 *

D

siTRIM29

0.25 Absorbance at 595 nm

0.2 0.15 0.1 0.05 0

HeLa

SiHa

E

siCTRL

HeLa S3

*

*

1.2 1

HeLa

SiHa

siCTRL

siTRIM29

1.4

0.8

siTRIM29

217

U

210 211

Relative absorbance at 595 nm

208 209

219

F

2.10. Analysis of focal adhesion area

3.1. TRIM29 regulates cell adhesion and invasion

O

206

218

R O

204 205

3. Results

P

202 203

buffer overnight at 4 °C and incubated with goat Alexa Fluor 488labeled anti-mouse IgG or goat Alexa Fluor 555-labeled anti-rabbit IgG antibody (Invitrogen Life Technologies, Carlsbad, CA) for 1 h at room temperature. DNA was counterstained with 0.4 μg/ml DAPI in PBS for 30 min at room temperature. Confocal images were taken using an upright microscope (FV1000, Olympus, Tokyo, Japan).

Absorbance at 595 nm

200 201

3

0.6 0.4 0.2 0 HeLa S3 HeLa

SiHa

Fig. 1. TRIM29 regulates cell adhesion and invasion. (A,B,C) Effect of TRIM29 knockdown on cell adhesion. HeLa S3 (A), HeLa (B) and SiHa (C) cells transfected with siCTRL or siTRIM29 were incubated on wells coated with fibronectin, fibrinogen, collagen I, collagen IV and laminin I for 1 h. Cells that had adhered to basement membrane proteins were stained and extracted to measure absorbance at 595 nm. Standard deviations of each result were calculated from three independent experiments. P values were determined by Student's t test (*, P b 0.05). Error bars show s.d. (D) Effect of TRIM29 knockdown on invasion to the basement membrane matrix. HeLa S3, HeLa and SiHa cells transfected with siCTRL or siTRIM29 were incubated on membranes coated with basement membrane matrix for 1 h. Invasive cells were stained and extracted to measure absorbance at 595 nm. Standard deviations of each result were calculated from three independent experiments. P values were determined by Student's t test (*, P b 0.05). Error bars show s.d. (E) Effect of TRIM29 depletion on cell–cell interactions. HeLa S3, HeLa and SiHa cells were transfected with siCTRL and siTRIM29 and then incubated for 48 h.

Please cite this article as: Y. Masuda, et al., TRIM29 regulates the p63-mediated pathway in cervical cancer cells, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbamcr.2015.05.035

222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238

Y. Masuda et al. / Biochimica et Biophysica Acta xxx (2015) xxx–xxx

3.2. Effect of TRIM29 knockdown on focal adhesion

243 244

ITGB1 plays a major role in binding to ECM components and contributes to the formation of focal adhesion [21]. To examine the effect of TRIM29 depletion on focal adhesion area of cells, we depleted HeLa S3, HeLa and SiHa cells of TRIM29 by siRNA transfection and then incubated the TRIM29-depleted cells on fibronectin-coated coverslips in serum-free medium for 24 h (Fig. 2A and B). We defined the ratio of ITGB1-stained area to DAPI-stained area as focal adhesion area. TRIM29 knockdown increased the focal adhesion area in HeLa S3 and HeLa cells but not in SiHa cells (Fig. 2C). These findings suggest that TRIM29 regulates focal adhesion depending on the type of cells.

A

HeLa S3

254 255

siTRIM29

siCTRL

SiHa

siTRIM29

siCTRL

siTRIM29

P

siCTRL

To reveal the relationship between TRIM29 and the behavior of cervical cancer cells, we examined the effects of TRIM29 knockdown on relative mRNA levels of p63 and EMT-related transcription factors. We also examined E2F and HOXB transcription factors, which are associated with cancer progression [22,23]. We suppressed TRIM29 in HeLa S3, HeLa and SiHa cells by siRNA transfection and analyzed the expression of indicated genes by qRT-PCR. Among the indicated genes, TRIM29 knockdown decreased the mRNA level of p63, whereas the mRNA level of ZEB1 was elevated in TRIM29-depleted cells (Fig. 3A–C). We also found that p53 and SNAIL2 were significantly upregulated depending on the cell type (Fig. 3A–C). The p63 gene has two distinct promoters that produce two subclasses of p63: transactivating p63 (TAp63) and N-terminal truncated p63 (ΔNp63) [24]. There are also C-terminal splicing variants

HeLa

E

D

ITGB1

C

T

Actin

R

E

DAPI

R

Merge

O

B

HeLa S3 HeLa + siCTRL: + - + siTRIM29: - + TRIM29 Actin

C

siCTRL

SiHa + - +

*

*

HeLa S3

HeLa

siTRIM29

3.5 Focal adhesion area

251 252

C

249 250

N

247 248

U

245 246

253

F

242

3.3. TRIM29 transcriptionally regulates ZEB1 and TAp63α isoform

O

241

in HeLa and SiHa cells (Fig. 1E). These findings suggest that TRIM29 regulates adhesion and invasion depending on the type of cervical cancer cells.

239 240

R O

4

3 2.5 2 1.5 1 0.5 0 SiHa

Fig. 2. TRIM29 regulates focal adhesion. (A) Effect of TRIM29 knockdown on focal adhesion. HeLa S3, HeLa and SiHa cells were transfected with siCTRL or siTRIM29 and incubated on fibronectin-coated coverslips in a serum-free medium. After 48 h, cells were fixed and stained with anti-actin and anti-ITGB1 antibodies. DNA was counterstained with DAPI. Scale bar, 40 μm. (B) Immunoblot analysis to confirm knockdown of TRIM29. HeLa S3, HeLa and SiHa cells were transfected with siCTRL or siTRIM29. After 48 h, whole cell lysates of TRIM29-depleted cells were subjected to SDS-PAGE and analyzed by immunoblotting. Actin was used as a loading control. (C) Quantification of focal cell adhesion area. Focal cell adhesion area was calculated as the ratio of ITGB1-stained area to DAPI-stained. ITGB1- and DAPI-stained areas were quantified using NIH ImageJ software (n = 20). P values were determined by Student's t test (*, P b 0.05). Error bars show s.d.

Please cite this article as: Y. Masuda, et al., TRIM29 regulates the p63-mediated pathway in cervical cancer cells, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbamcr.2015.05.035

256 257 258 259 260 261 262 263 264 265 266 267

Y. Masuda et al. / Biochimica et Biophysica Acta xxx (2015) xxx–xxx

5

A siCTRL siTRIM29

HeLa S3

1.5 1 0.5

O

*

D HeLa

T

0.00018

siCTRL siTRIM29 *

0.00008

0.00015

R

TA

p6 N 3 p6 p6 3 3 p6 3 p6 3

N C O

0

*

0.0001

0.00008 0.00006

*

0.00004

0.00006 0.00005 0.00004 0.00003 0.00002

0.00002

0.00001

0

0

*

p6 N 3 p6 p6 3 3 p6 3 p6 b 3

R

0.0002

*

0.00012

p6 N 3 p6 p6 3 3 p6 3 p6 3

0.00025

*

0.00007

0.00014

E

0.0003

Relative mRNA level

0.00035

SiHa siCTRL siTRIM29

0.00016

0.00005

*

TA

*

C

*

0.0004

0.0001

*

F

E

E

siCTRL siTRIM29

Relative mRNA level

2

0

HeLa S3

0.00045

2.5

*

R O

*

siCTRL siTRIM29

P

*

SiHa

3 Relative mRNA level

siCTRL siTRIM29

*

TA

Relative mRNA level

*

F

C

HeLa 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

*

*

*

Relative mRNA level

Relative mRNA level

*

B

D

*

*

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

U

Fig. 3. TRIM29 regulates the expression of TAp63α and EMT-related genes. HeLa S3 (A and D), HeLa (B and E) and SiHa (C and F) cells were transfected with siCTRL and siTRIM29 and then incubated for 48 h. The relative mRNA levels of TRIM29, p63 and the indicated genes were analyzed by quantitative RT-PCR. Standard deviations of each result were calculated from three independent experiments. P values were determined by Student's t test (*, P b 0.05). Error bars show s.d.

268 269 270 271 272 273 274 275 276 277 278 279

known as p63α, p63β and p63γ. To determine which isoforms of p63 are regulated by TRIM29, we performed qRT-PCR using previously reported primers that are specific for the isoforms of p63 (TAp63, ΔNp63, p63α, p63β and p63γ) [3]. While TAp63 isoforms were highly expressed, mRNA level of the ΔNp63 isoform was much lower than that of the TAp63 isoform (Fig. 3D–F). In addition, mRNA level of the p63α isoform was much higher than the levels of p63β and p63γ isoforms (Fig. 3D–F). TRIM29 knockdown in each cell line suppressed the expression of TAp63, p63α and p63γ isoforms (Fig. 3D–F). Taken together, these findings indicate that TRIM29 is a transcriptional regulator of TAp63α and ZEB1 in cervical cancer cells.

3.4. TRIM29 regulates the expression of cell adhesion genes

280

p63 regulates the expression of cell adhesion genes including β1integrin (ITGB1), β4-integrin (ITGB4), α3-integrin (ITGA3), α5integrin (ITGA5), α6-integrin (ITGA6), fibronectin (FN1) and vimentin (VIM) [2]. To clarify the relationship between TRIM29 and p63, we analyzed the effect of TRIM29 depletion on the expression of these genes. Knockdown of TRIM29 in HeLa S3 and HeLa cells had similar effects on the expression of ITGB1, ITGB4, ITGA5 and FN1 (Fig. 4A and B). TRIM29 knockdown in SiHa cells caused an increase in the expression of ITGB4 and a decrease in the expression of FN1 (Fig. 4C). In addition,

281

Please cite this article as: Y. Masuda, et al., TRIM29 regulates the p63-mediated pathway in cervical cancer cells, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbamcr.2015.05.035

282 283 284 285 286 287 288 289

6

Y. Masuda et al. / Biochimica et Biophysica Acta xxx (2015) xxx–xxx

A Relative mRNA level

0.3

siCTRL

HeLa S3

0.25 0.2

expression of ZEB1 in HeLa S3, HeLa and SiHa cells (Fig. 5A). While ITGB1 was increased in HeLa S3 and HeLa cells by TRIM29 knockdown, ITGB4 was decreased in HeLa cells and was slightly increased in SiHa cells by TRIM29 depletion (Fig. 5A). Nuclear localization of p63 is required for its function as a transcription factor. To examine whether TRIM29 regulates the subcellular localization of p63, we incubated TRIM29-depleted cells on coverslips and then analyzed the subcellular localization of p63 by immunofluorescence staining. TRIM29 depletion did not alter the subcellular localization of p63 in each cell line (Fig. 5B). Depletion of TRIM29 elevated the level of ZEB1 protein in each cell line (Fig. 5A). These findings raised the possibility that TRIM29 suppresses the expression of ZEB1 by interacting with TAp63α in cervical cancer cells. To determine whether TRIM29 physically interacts with TAp63α, we generated HeLa S3 cells stably expressing N-terminal FLAG-tagged TRIM29, prepared nuclear extracts from the cells, and purified FLAG-tagged TRIM29-binding proteins by anti-FLAG immunoprecipitation. As expected, we found that TRIM29 interacted with TAp63α (Fig. 5C). A recent study showed that knockdown of MED12, which is a key subunit of the Mediator complex, upregulates the expression of EMT-related genes [25]. To further determine the role of TRIM29 in the transcription of EMT-related genes, we confirmed whether TRIM29 interacts with MED12 and RNA polymerase II (Pol II). Western blot analysis showed that TRIM29 binds to Pol II and MED12 (Fig. 5C). These findings suggest that TRIM29 regulates ZEB1 suppression and integrin expression through TAp63α, Pol II and MED12.

*

siTRIM29

*

0.15 0.1

*

*

*

0.05 0 VIM

F

ITGB1 ITGB4 ITGA3 ITGA5 ITGA6 FN1

0.4

HeLa

siCTRL siTRIM29

0.25

R O

0.3

*

0.2 0.15 0.1

*

*

P

Relative mRNA level

0.35

O

B

0.05 0 VIM

D

ITGB1 ITGB4 ITGA3 ITGA5 ITGA6 FN1

C

E

3.6. Forced expression of TRIM29 upregulates ITGB4

SiHa

siCTRL

0.25

C

0.2 0.15

*

0.05

E

0.1

*

0

VIM

R

ITGB1 ITGB4 ITGA3 ITGA5 ITGA6 FN1

290

N

C

O

Fig. 4. Effect of TRIM29 knockdown on expression of integrins, fibronectin and vimentin. Quantitative RT-PCR analysis was performed to measure mRNA levels of integrins, FN1 and VIM in TRIM29-knockdown HeLa S3 (A), HeLa (B) and SiHa (C) cells. Using the same procedure as that described in the legend of Fig. 3, mRNA levels of ITGB1, ITGB4, ITGA5, ITGA6, FN1 and VIM were analyzed by quantitative RT-PCR. Standard deviations of each result were calculated from three independent experiments. P values were determined by Student's t test (*, P b 0.05). Error bars show s.d.

295

3.5. TRIM29 interacts with TAp63α in cervical cancer cells

296

To confirm that TRIM29 regulates the p63-mediated pathway, we examined the effect of TRIM29 knockdown on the protein levels of p63, ZEB1, ITGB1 and ITGB4 by immunoblot analysis. HeLa S3, HeLa and SiHa cells were transfected with TRIM29 siRNA, and the whole cell lysates were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. TRIM29 knockdown led to a slight decrease in the protein level of TAp63α in HeLa S3 cells but not in HeLa and SiHa cells (Fig. 5A). TRIM29 knockdown caused increased

291 292

297 298 299 300 301 302 303

U

293 294

loss of TRIM29 in each cell line increased the mRNA level of VIM, which is a marker of mesenchymal cells (Fig. 4). These findings suggest that TRIM29 regulates the expression of cell adhesion molecules in cervical cancer cells. However, the transcriptional regulation of cell adhesion genes by TRIM29 is dependent on the type of cells.

306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331

To address the reciprocal relationship between TRIM29 and TAp63α, we analyzed the effect of TRIM29 overexpression on the expression of p63 isoforms and integrins by qRT-PCR. We confirmed that the mRNA level of TRIM29 was greatly increased in FLAG-TRIM29-expressing HeLa S3 cells (Fig. 6A). TRIM29 overexpression caused an increase in the expression levels of TAp63, p63α, p63β and p63γ but not in that of ΔNp63 (Fig. 6B). Overexpression of TRIM29 also resulted in increases in ITGB4, ITGA3 and ITGA6 expression levels (Fig. 6C). Although cells expressing FLAG-TRIM29 showed an increase in ITGB1 expression, TRIM29 overexpression had less of an impact on ITGB1 expression than did TRIM29 knockdown (Figs. 4A and 6C). We also confirmed the effect of TRIM29 overexpression on protein levels of TAp63α, ITGB1 and ITGB4 by Western blotting. TRIM29 overexpression resulted in an increased level of ITGB4 but not in increased levels of TAp63α and ITGB1 (Fig. 6D). To confirm the effect of TRIM29 overexpression on cell invasion and adhesion, we performed in vitro cell invasion and adhesion assays. TRIM29 overexpression caused a 1.9-fold increase in the invasion acivity of HeLa S3 cells (Fig. 6E). Although TRIM29 overexpression decreased the adhesion activity of cells to collagen IV, there was no significant difference in the adhesion activity of cells to other ECM components (Fig. 6F). HeLa S3 cells expressing FLAG-TRIM29 also exhibited a marked change with tight cobblestone-like structures in cell morphology (Fig. 6G). These findings suggest that TRIM29 overexpression enhances cell invasion and cell–cell interactions in HeLa S3 cells.

332 333

3.7. TRIM29 is required for regulation of the p63-mediated pathway

357

To clarify the functional relationship between TRIM29 and TAp63α, we generated HeLa S3 cells stably expressing FLAG-tagged TAp63α and knocked down TRIM29 in the cells by siRNA transfection. We confirmed that FLAG-tagged TAp63α was expressed at a higher level than that of endogenous TAp63α (Fig. 7A and B). Although TRIM29 depletion increased the levels of ZEB1 and ITGB1, overexpression of TAp63α did not suppress the elevation of ZEB1 and ITGB1 levels (Fig. 7A and B).

358 359

T

siTRIM29

R

Relative mRNA level

0.3

304 305

Please cite this article as: Y. Masuda, et al., TRIM29 regulates the p63-mediated pathway in cervical cancer cells, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbamcr.2015.05.035

334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356

360 361 362 363 364

Y. Masuda et al. / Biochimica et Biophysica Acta xxx (2015) xxx–xxx

A

C

IP: FLAG Input

Control FLAG-TRIM

TRIM29 TAp63

29

29

SiHa + - +

Control FLAG-TRIM

HeLa S3 HeLa + siCTRL: + - + siTRIM29: - +

7

ZEB1

FLAG

ITGB1 TAp63

ITGB4

Pol II

GAPDH

MED12

F

Actin

siCTRL

HeLa

siTRIM29

siCTRL

SiHa

siTRIM29

siTRIM29

P

p63

siCTRL

R O

HeLa S3

O

B

E

D

Actin

C

T

DAPI

R

E

Merge

N C O

R

Fig. 5. TRIM29 suppresses ZEB1 expression through interaction with TAp63α. (A) Effect of TRIM29 knockdown on protein levels of TRIM29, TAp63α, ZEB1, ITGB1 and ITGB4 in HeLa S3, HeLa and SiHa cells. HeLa S3, HeLa and SiHa cells were transfected with siCTRL and siTRIM29 and then incubated for 48 h. Cell lysates were subjected to SDS-PAGE. Protein levels of TRIM29, p63, ZEB1, ITGB1 and ITGB4 were analyzed by immunoblotting. Actin was used as a loading control. (B) Effect of TRIM29 knockdown on subcellular localization of p63. HeLa S3, HeLa and SiHa cells were transfected with siCTRL or siTRIM29 and incubated on coverslips. After 48 h, cells were fixed and stained with anti-actin and anti-p63 antibodies. DNA was counterstained with DAPI. Scale bar, 40 μm. (C) Western blot analysis of proteins associated with FLAG-tagged TRIM29. M2-agarose was incubated with nuclear extracts of HeLa S3 cells stably expressing FLAG-tagged TRIM29. The bound proteins were washed, eluted by 3× FLAG peptides, and then analyzed by Western blotting. Cells that were not transfected were used as control cells.

377

4. Discussion

378 379

In this study, we showed that TRIM29 is an important regulator of the p63-mediated pathway in cervical cancer cells (Fig. 7D). p63 has a critical role in cell adhesion through the regulation of integrin expression [2], and it inhibits the expression of ZEB1 in pancreatic and bladder cancer cells [30,31]. TRIM29 knockdown increased ZEB1 and ITGB1

366 367 368 369 370 371 372 373 374 375

380 381 382

U

376

These findings suggest that TRIM29 regulates the expression of ZEB1 and ITGB1 independently of TAp63α. We showed that TRIM29 depletion increases the adhesion activity of HeLa S3 cells to ECM components (Fig. 1A). To examine the effect of TAp63α overexpression on cell adhesion to the ECM, we performed in vitro cell adhesion assays. In the presence of endogenous TRIM29, overexpression of TAp63α had no effect on the adhesion activity of cells to ECM components (Fig. 7C). Conversely, overexpression of TAp63α increased the adhesion activity of TRIM29-depleted cells to fibronectin, collagen I and collagen IV (Fig. 7C). These findings suggest that TRIM29 negatively regulates an increase in the adhesion activity of HeLa S3 cells by TAp63α.

365

levels in cervical cancer cells. However, TAp63α overexpression did not suppress the elevation of ZEB1 and ITGB1 levels in TRIM29depleted cells, indicating that TRIM29 suppresses the expression of ZEB1 and ITGB1 independently of TAp63α. We also identified MED12, which is a key component of the Mediator complex [28,29], as a binding partner of TRIM29. Given that loss of MED12 upregulates EMT-related genes in lung cancer cells [25], our observations raise the possibility that TRIM29 suppresses the expression of ZEB1 and ITGB1 through MED12. Furthermore, knockdown and overexpression experiments showed that TRIM29 regulates the mRNA level of TAp63α, suggesting that mRNA levels of TAp63α are regulated through the interaction with TRIM29 and MED12. This is important for understanding the detailed mechanism by which TRIM29 regulates TAp63α via MED12. In cervical cancer cells, depletion and overexpression of TRIM29 affected the expression pattern of ITGB1 and ITGB4 and adhesion activity to the ECM. ITGB1 is essential for interaction of basal epithelial cells with the ECM [32]. ITGB4 expression disrupts adhesive activity and enhances invasive activity of epithelial cells [33]. However, loss of TRIM29 exerted different effects on integrin expression and cell adhesion in HPV-18positive cells (HeLa and HeLa S3) and HPV-16-positive cells (SiHa). In

Please cite this article as: Y. Masuda, et al., TRIM29 regulates the p63-mediated pathway in cervical cancer cells, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbamcr.2015.05.035

383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402

8

Y. Masuda et al. / Biochimica et Biophysica Acta xxx (2015) xxx–xxx

A

*

0.0004 0.00035

1.25

Relative mRNA level

Relative mRNA level

B

*

1.5

1 0.75 0.5 0.25

0.0003

CTRL FLAG-TRIM29

*

0.00025 0.0002 0.00015

*

0.0001

*

0.00005

0

AG C -T TR R L IM 29

0 Np63 p63

p63

p63

D

0.12 0.1

*

0.08 0.04

*

*

0

E

ITGB1 ITGB4 ITGA3 ITGA5 ITGA6

F

CTRL

T

E

0.1

0.4 0.35 0.3 0.25

0.08 0.07 0.06

0.15

G

CTRL

FLAG-TRIM29

*

0.05 0.04

R

0.2

0.09

C

Absorbance at 595 nm

0.45

E

*

FLAG TAp63 ITGB1 ITGB4 GAPDH

P

0.06 0.02

Absorbance at 595 nm

Control FLAG-TRIM 29

FLAG-TRIM29

R O

CTRL

0.14

D

Relative mRNA level

* 0.16

O

C

F

FL

TAp63

FLAG-TRIM29

0.03 0.02 0.01 0

C

FL

AG

O

C -T TR R L IM 29

0

R

0.1 0.05

U

N

Fig. 6. Effect of overexpression of TRIM29 on p63 and integrin expression. (A–C) Quantitative PCR analysis of the expression levels of TRIM29 (A), p63 isoforms (B) and integrins (C) in HeLa S3 cells overexpressing TRIM29. P values were determined by Student's t test (*, P b 0.05). Error bars show s.d. (D) Effect of TRIM29 overexpression on protein levels of p63, ITGB1 and ITGB4 in HeLa S3. Cell lysates from HeLa S3 cells expressing FLAG-tagged TRIM29 were subjected to SDS-PAGE. Protein levels of p63, ITGB1 and ITGB4 were analyzed by immunoblotting. (E) Effect of TRIM29 overexpression on cell invasion to the basement membrane matrix. HeLa S3 cells stably expressing FLAG-tagged TRIM29 were incubated on membranes coated with basement membrane matrix for 1 h. Invasive cells were stained and extracted to measure absorbance at 595 nm. Standard deviations of each result were calculated from three independent experiments. P values were determined by Student's t test (*, P b 0.05). Error bars show s.d. (F) Effect of TRIM29 overexpression on cell adhesion to ECM proteins. HeLa S3 cells stably expressing FLAG-tagged TRIM29 were incubated in wells coated with fibronectin, fibrinogen, collagen I, collagen IV and laminin I for 1 h. Cells that had adhered to basement membrane proteins were stained and extracted to measure absorbance at 595 nm. Standard deviations of each result were calculated from three independent experiments. P values were determined by Student's t test (*, P b 0.05). Error bars show s.d. (G) Effect of TRIM29 overexpression on cell–cell interactions. Images of HeLa S3 cells expressing FLAG-tagged TRIM29 are shown.

403 404 405 406 407 408 409

cervical cancer cells, HPV genomes are integrated into the host genome, followed by chromosomal rearrangement and instability [34]. The integration of HPV genomes into the host genome disrupts several genes [35,36]. Previous studies showed that HPV-18 and HPV-16 genomes are integrated into chromosomes 8 and 13, respectively [35,36]. Thus, the difference in HPV integration sites may cause an alteration in the expression of cell adhesion genes. Further detailed studies on the

mechanism by which TRIM29 regulates the expression of EMT-related genes in HPV-18 and HPV-16-positive cells might be useful for understanding the phenotypes of cervical cancers. In this study, we found that TRIM29 enhances invasion of cervical cancer cells. While TRIM29 inhibits invasion of breast cancer cells [14], it enhances invasion of pancreatic and lung cancer cells [15,16]. Degradation of the ECM by MMPs has an important role in cell invasion and

Please cite this article as: Y. Masuda, et al., TRIM29 regulates the p63-mediated pathway in cervical cancer cells, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbamcr.2015.05.035

410 411 412 413 414 415 416

Y. Masuda et al. / Biochimica et Biophysica Acta xxx (2015) xxx–xxx

0.04 0.03 0.02 0.01 0

*

0.5 0.4 0.3 0.2 0.1 0

TRIM29

0.003 0.0025 0.002 0.0015 0.001 0.0005 0

TAp63

ZEB1

0.06 0.04 0.02 0

ITGB1

F

0.05

0.6

siCTRL siCTRL + TAp63 -FLAG siTRIM29 siTRIM29 + TAp63 -FLAG 0.1 * 0.08

Relative mRNA level

*

Relative mRNA level

0.06

Relative mRNA level

C

B HeLa S3

ZEB1 ITGB1

0.12 0.1 0.08 0.06 0.04 0.02 0

*

*

E

D

GAPDH

siCTRL siCTRL + TAp63 -FLAG siTRIM29 siTRIM29 + TAp63 -FLAG

R O

endo TAp63

TRIM29

0.14

P

TAp63

TAp63 -FLAG

Absorbance at 595 nm

*

TAp63 -FLAG: - + - + siCTRL: + + - siTRIM29: - - + + FLAG

O

Relative mRNA level

A

9

T

D TAp63

C

Suppression of ZEB1 Integrin expression

E

TRIM29

Change in cell adhesion

419 420 421 422 423 424 425 426 427 428 429 430 431 432 433

metastasis [37]. It has been reported that HPV infection increases the expression of various MMPs in cervical cancer cells [8]. Thus, the invasive activity of cervical cancer cells may be regulated by the interplay between the function of TRIM29 and the effect of HPV infection. Further studies will be required for determination of the mechanism by which TRIM29 regulates the invasive activity of cervical cancer cells. We found that TAp63α is maintained at a constant protein level independently of the mRNA level of TAp63α. There are several reports that ubiquitination of TAp63α by E3 ubiquitin ligase plays an important role in its stability. Ectopically expressed TAp63α is ubiquitinated and degraded in an Itch-dependent manner [26]. In contrast, it has been reported that βTrCP1 ubiquitinates and stabilizes TAp63α [27]. These findings suggest that TAp63α is maintained at a constant level in a ubiquitination-dependent manner specific for cervical cancer cells. Our results demonstrate that TRIM29 regulates the p63-mediated pathway and the behavior of cervical cancer cells. Further studies on the mechanism by which TRIM29 regulates the characteristics of

U

417 418

N C O

R

R

Fig. 7. Functional relationship between TRIM29 and TAp63α. (A) Quantitative PCR analysis of the expression levels of TRIM29, TAp63, ZEB1 and ITGB1 in HeLa S3 cells overexpressing TAp63α. HeLa S3 cells stably expressing FLAG-tagged TAp63 were transfected with siCTRL and siTRIM29 and then incubated for 48 h. The relative mRNA levels of the indicated genes were analyzed by quantitative RT-PCR. Standard deviations of each result were calculated from three independent experiments. P values were determined by Student's t test (*, P b 0.05). Error bars show s.d. (B) Effect of TRIM29 knockdown on cells overexpressing TAp63α. HeLa S3 cells expressing FLAG-tagged TAp63 were depleted of TRIM29 by siRNA transfection. Cell lysates from the cells were subjected to SDS-PAGE followed by Western blotting with the indicated antibodies. GAPDH was used as a loading control. (C) Effect of TAp63α overexpression on cell adhesion to ECM proteins. Cells were incubated in wells coated with fibronectin, fibrinogen, collagen I, collagen IV and laminin I for 1 h. The cells that had adhered to basement membrane proteins were stained and extracted to measure absorbance at 595 nm. Standard deviations of each result were calculated from three independent experiments. P values were determined by Student's t test (*, P b 0.05). Error bars show s.d. (D) Schematic representation of a putative role of TRIM29 in the p63-mediated pathway. TRIM29 binds to TAp63α in cervical cancer cells and regulates the expression of ZEB1 and integrins.

cervical cancer cells may contribute to the establishment of new thera- 434 pies for cervical cancers. 435 Conflict of interest The authors declare no competing financial interests.

436 437

Acknowledgments

438

We would like to thank Drs. Toshio Kitamura (Tokyo University) and Kentaro Hanada (National Institute of Infectious Diseases) for the plasmids. We are grateful to Ms. Miho Uchiumi for help in preparing the manuscript and Ms. Misumi Matsuo for technical assistance. This work was supported in part by KAKENHI (24112006, 2439 0065) from the Ministry of Education, Culture, Sports, Science and Technology in Japan and by the Naito Foundation and the Uehara Memorial Foundation (to S. Hatakeyama).

439

Please cite this article as: Y. Masuda, et al., TRIM29 regulates the p63-mediated pathway in cervical cancer cells, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbamcr.2015.05.035

440 441 442 443 444 445 446 Q4

Y. Masuda et al. / Biochimica et Biophysica Acta xxx (2015) xxx–xxx

F

O

R O

[1] S. Lamouille, J. Xu, R. Derynck, Molecular mechanisms of epithelial–mesenchymal transition, Nat. Rev. Mol. Cell Biol. 15 (2014) 178–196. [2] D.K. Carroll, J.S. Carroll, C.O. Leong, F. Cheng, M. Brown, A.A. Mills, J.S. Brugge, L.W. Ellisen, p63 regulates an adhesion programme and cell survival in epithelial cells, Nat. Cell Biol. 8 (2006) 551–561. [3] X. Su, D. Chakravarti, E.R. Flores, p63 steps into the limelight: crucial roles in the suppression of tumorigenesis and metastasis, Nat. Rev. Cancer 13 (2013) 136–143. [4] M.H. Forouzanfar, K.J. Foreman, A.M. Delossantos, R. Lozano, A.D. Lopez, C.J. Murray, M. Naghavi, Breast and cervical cancer in 187 countries between 1980 and 2010: a systematic analysis, Lancet 378 (2011) 1461–1484. [5] J.M. Walboomers, M.V. Jacobs, M.M. Manos, F.X. Bosch, J.A. Kummer, K.V. Shah, P.J. Snijders, J. Peto, C.J. Meijer, N. Munoz, Human papillomavirus is a necessary cause of invasive cervical cancer worldwide, J. Pathol. 189 (1999) 12–19. [6] G.M. Clifford, J.S. Smith, M. Plummer, N. Munoz, S. Franceschi, Human papillomavirus types in invasive cervical cancer worldwide: a meta-analysis, Br. J. Cancer 88 (2003) 63–73. [7] N. Munoz, F.X. Bosch, S. de Sanjose, R. Herrero, X. Castellsague, K.V. Shah, P.J. Snijders, C.J. Meijer, G. International Agency for Research on Cancer Multicenter Cervical Cancer Study, Epidemiologic classification of human papillomavirus types associated with cervical cancer, N. Engl. J. Med. 348 (2003) 518–527. [8] J. Kaewprag, W. Umnajvijit, J. Ngamkham, M. Ponglikitmongkol, HPV16 oncoproteins promote cervical cancer invasiveness by upregulating specific matrix metalloproteinases, PLoS One 8 (2013) e71611. [9] M.Y. Lee, C.Y. Chou, M.J. Tang, M.R. Shen, Epithelial–mesenchymal transition in cervical cancer: correlation with tumor progression, epidermal growth factor receptor overexpression, and snail up-regulation, Clin. Cancer Res. 14 (2008) 4743–4750. [10] G. Maity, S. Fahreen, A. Banerji, P. Roy Choudhury, T. Sen, A. Dutta, A. Chatterjee, Fibronectin–integrin mediated signaling in human cervical cancer cells (SiHa), Mol. Cell. Biochem. 336 (2010) 65–74. [11] L.N. Kapp, R.B. Painter, L.C. Yu, N. van Loon, C.W. Richard III, M.R. James, D.R. Cox, J.P. Murnane, Cloning of a candidate gene for ataxia-telangiectasia group D, Am. J. Hum. Genet. 51 (1992) 45–54. [12] S. Hatakeyama, TRIM proteins and cancer, Nat. Rev. Cancer 11 (2011) 792–804. [13] Y. Kanno, M. Watanabe, T. Kimura, K. Nonomura, S. Tanaka, S. Hatakeyama, TRIM29 as a novel prostate basal cell marker for diagnosis of prostate cancer, Acta Histochem. 116 (2014) 708–712. [14] L. Ai, W.J. Kim, M. Alpay, M. Tang, C.E. Pardo, S. Hatakeyama, W.S. May, M.P. Kladde, C.D. Heldermon, E.M. Siegel, K.D. Brown, TRIM29 suppresses TWIST1 and invasive breast cancer behavior, Cancer Res. 74 (2014) 4875–4887. [15] Z.P. Tang, Q.Z. Cui, Q.Z. Dong, K. Xu, E.H. Wang, Ataxia-telangiectasia group D complementing gene (ATDC) upregulates matrix metalloproteinase 9 (MMP-9) to promote lung cancer cell invasion by activating ERK and JNK pathways, Tumour Biol. 34 (2013) 2835–2842. [16] L. Wang, H. Yang, E.V. Abel, G.M. Ney, P.L. Palmbos, F. Bednar, Y. Zhang, J. Leflein, M. Waghray, S. Owens, J.E. Wilkinson, J. Prasad, M. Ljungman, A.D. Rhim, M. Pasca di Magliano, D.M. Simeone, ATDC induces an invasive switch in KRAS-induced pancreatic tumorigenesis, Genes Dev. 29 (2015) 171–183. [17] L.M. Albritton, L. Tseng, D. Scadden, J.M. Cunningham, A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection, Cell 57 (1989) 659–666. [18] S. Morita, T. Kojima, T. Kitamura, Plat-E: an efficient and stable system for transient packaging of retroviruses, Gene Ther. 7 (2000) 1063–1066. [19] J.D. Dignam, R.M. Lebovitz, R.G. Roeder, Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei, Nucleic Acids Res. 11 (1983) 1475–1489.

P

448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 Q5 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 558

[20] H. Takahashi, S. Martin-Brown, M.P. Washburn, L. Florens, J.W. Conaway, R.C. Conaway, Proteomics reveals a physical and functional link between hepatocyte nuclear factor 4alpha and transcription factor IID, J. Biol. Chem. 284 (2009) 32405–32412. [21] A. Huttenlocher, A.R. Horwitz, Integrins in cell migration, Cold Spring Harb. Perspect. Biol. 3 (2011) a005074. [22] H.Z. Chen, S.Y. Tsai, G. Leone, Emerging roles of E2Fs in cancer: an exit from cell cycle control, Nat. Rev. Cancer 9 (2009) 785–797. [23] N. Shah, S. Sukumar, The Hox genes and their roles in oncogenesis, Nat. Rev. Cancer 10 (2010) 361–371. [24] A. Yang, M. Kaghad, Y. Wang, E. Gillett, M.D. Fleming, V. Dotsch, N.C. Andrews, D. Caput, F. McKeon, p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities, Mol. Cell 2 (1998) 305–316. [25] S. Huang, M. Holzel, T. Knijnenburg, A. Schlicker, P. Roepman, U. McDermott, M. Garnett, W. Grernrum, C. Sun, A. Prahallad, F.H. Groenendijk, L. Mittempergher, W. Nijkamp, J. Neefjes, R. Salazar, P. Ten Dijke, H. Uramoto, F. Tanaka, R.L. Beijersbergen, L.F. Wessels, R. Bernards, MED12 controls the response to multiple cancer drugs through regulation of TGF-beta receptor signaling, Cell 151 (2012) 937–950. [26] M. Rossi, R.I. Aqeilan, M. Neale, E. Candi, P. Salomoni, R.A. Knight, C.M. Croce, G. Melino, The E3 ubiquitin ligase Itch controls the protein stability of p63, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 12753–12758. [27] J.R. Gallegos, J. Litersky, H. Lee, Y. Sun, K. Nakayama, K. Nakayama, H. Lu, SCF TrCP1 activates and ubiquitylates TAp63gamma, J. Biol. Chem. 283 (2008) 66–75. [28] R.C. Conaway, J.W. Conaway, The Mediator complex and transcription elongation, Biochim. Biophys. Acta 1829 (2013) 69–75. [29] D.J. Taatjes, The human Mediator complex: a versatile, genome-wide regulator of transcription, Trends Biochem. Sci. 35 (2010) 315–322. [30] M.N. Tran, W. Choi, M.F. Wszolek, N. Navai, I.L. Lee, G. Nitti, S. Wen, E.R. Flores, A. Siefker-Radtke, B. Czerniak, C. Dinney, M. Barton, D.J. McConkey, The p63 protein isoform DeltaNp63alpha inhibits epithelial–mesenchymal transition in human bladder cancer cells: role of MIR-205, J. Biol. Chem. 288 (2013) 3275–3288. [31] P. Tucci, M. Agostini, F. Grespi, E.K. Markert, A. Terrinoni, K.H. Vousden, P.A. Muller, V. Dotsch, S. Kehrloesser, B.S. Sayan, G. Giaccone, S.W. Lowe, N. Takahashi, P. Vandenabeele, R.A. Knight, A.J. Levine, G. Melino, Loss of p63 and its microRNA205 target results in enhanced cell migration and metastasis in prostate cancer, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 15312–15317. [32] I. Taddei, M.A. Deugnier, M.M. Faraldo, V. Petit, D. Bouvard, D. Medina, R. Fassler, J.P. Thiery, M.A. Glukhova, Beta1 integrin deletion from the basal compartment of the mammary epithelium affects stem cells, Nat. Cell Biol. 10 (2008) 716–722. [33] W. Guo, Y. Pylayeva, A. Pepe, T. Yoshioka, W.J. Muller, G. Inghirami, F.G. Giancotti, Beta 4 integrin amplifies ErbB2 signaling to promote mammary tumorigenesis, Cell 126 (2006) 489–502. [34] C.A. Moody, L.A. Laimins, Human papillomavirus oncoproteins: pathways to transformation, Nat. Rev. Cancer 10 (2010) 550–560. [35] A. Adey, J.N. Burton, J.O. Kitzman, J.B. Hiatt, A.P. Lewis, B.K. Martin, R. Qiu, C. Lee, J. Shendure, The haplotype-resolved genome and epigenome of the aneuploid HeLa cancer cell line, Nature 500 (2013) 207–211. [36] K. Akagi, J. Li, T.R. Broutian, H. Padilla-Nash, W. Xiao, B. Jiang, J.W. Rocco, T.N. Teknos, B. Kumar, D. Wangsa, D. He, T. Ried, D.E. Symer, M.L. Gillison, Genome-wide analysis of HPV integration in human cancers reveals recurrent, focal genomic instability, Genome Res. 24 (2014) 185–199. [37] K. Kessenbrock, V. Plaks, Z. Werb, Matrix metalloproteinases: regulators of the tumor microenvironment, Cell 141 (2010) 52–67.

D

References

U

N

C

O

R

R

E

C

T

447

E

10

Please cite this article as: Y. Masuda, et al., TRIM29 regulates the p63-mediated pathway in cervical cancer cells, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbamcr.2015.05.035

503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557