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Introduction

The tumor protein D52 (TPD52) protein family consists of TPD52 (1), -53 (1–4), -54 (2,4), and -55 (5). The first identified protein of this family, TPD52, was found to be overexpressed in breast and lung cancers (6,7). Other family members have also been reported to be highly expressed in colon (8,9), ovary (10–12), testis (5,13), prostate (14), and breast (15–17) cancers. Previous reports showed that overexpression of tpd52 in non-malignant 3T3 fibroblasts induces malignant transformation and increases cell proliferation and anchorage-independent growth (18,19). Moreover, overexpression of tpd52 led to an increase in cell proliferation as well as phosphorylation of Akt/protein kinase B (PKB) in prostate cancer (20–22) and also protected the cells from apoptosis induced by androgen deprivation via activation of the Stat3/Bcl-2 pathway (21). In addition, TPD52 regulates cell migration and invasion (23) and inhibits DNA damage repair (24). TPD53 regulates the cell cycle and is highly upregulated at the G2-M phase transition (25). TPD52, -53, and -54 also interact with each other through their coiled-coil domains (4). They also have numerous other binding partners, such as MAL2 (26), the phospholipid binding protein Annexin VI (27), the SNARE protein (Synaptobrevin 2), a main components of the SNARE complex (28), 14–3–3 (29), and adipose differentiation-related protein (ADRP) (30). These reports strongly suggest that TPD52 family protein members are important candidate targets of molecular therapy (31).

Signaling pathways such as mitogen-activated protein kinase (MAPK), Akt/PKB, and integrin signaling regulate cell proliferation (32), survival (33), and metastasis (34). Moreover, cross-talk of integrin signaling with MAPK signaling (35) and Akt/PKB signaling (36,37) has been widely reported. Despite these findings, the direct effects of TPD52 family protein members on cell proliferation, migration, invasion, and metastasis in oral squamous cell carcinoma cell (OSCC) are still under investigation.

We recently reported (38) that TPD54 is highly expressed in both OSCC and in hyperplastic epidermal cells around cancer tissue. We also showed that TPD54 is a negative regulator of extracellular matrix (ECM)-dependent attachment and migration in OSCC (39). However, little is known concerning the detailed physiological and pathological functions of TPD54. Herein, as an extension of our previous study, we investigated in greater detail the expression of D52 proteins in OSCC tissue, and the effects of TPD52, -53 and -54 that underlie cell growth, migration, and invasion of OSCC cells. The results showed that TPD54 was overexpressed in both highly and poorly differentiated OSCC tissue, while the expression of TPD52 was not as high as that of TPD54. TPD53 was moderately expressed. Furthermore, TPD54 inhibited the colony formation and migration. These results indicated that overexpression of TPD54 negatively regulates tumor growth in vivo.

Materials and methods

Co-immunoprecipitation (Co-IP) analysis

Co-immunoprecipitation analysis was carried out using a commercial kit [Immunoprecipitation kit (Protein G), Sigma-Aldrich, St. Louis, MO, USA]. Subconfluent SAS cells in a 10-cm culture dish were lysed with the lysis buffer, and the total extract was immunoprecipitated with 5 µg of anti-TPD52, -TPD53, -TPD54 or -GAPDH antibodies, or 5 µg of pre-immune mouse IgG (described above), according to the manufacturer's protocol. Subsequently, the precipitated proteins were eluted in 1X SDS sample buffer (Bio-Rad, Hercules, CA, USA) containing 5% of 2-mercaptoethanol, and co-precipitating proteins were then analyzed by western blotting as described in another subsection.

Protein preparation and western blot analysis

Total cellular proteins were collected with Triton X-100 lysis buffer [50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 0.5% Triton X-100, 5 mM ethrylendiaminetetraacetic acid (EDTA), 1 mM sodium o-vanadate] supplemented with Complete Mini protease inhibitor tablet (Roche Diagnostics, Mannheim, Germany) 48 h after gene-transfection. Protein concentrations were measured using the Quick Start Bradford reagent (Bio-Rad) with bovine serum albumin (BSA) as a standard. For western blot analysis, 20 µg of total cellular protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 4–20% gradient gel (Bio-Rad) and blotted onto a polyvinylidene difluoride membrane using iBlot 2 (Thermo Fisher Scientific, Waltham, MA, USA). After blocking of non-specific binding with 0.2% non-fat dry milk (Cell Signaling Technology, Danvers, MA, USA) in Tris-buffered saline (Takara Bio, Shiga, Japan), the membrane was incubated with the following primary antibodies: anti-TPD52 [1/1,000 dilution; rabbit monoclonal antibody, Abcam, Branford, CT, USA (ab182578)]; anti-TPD53 [1/1,000 dilution; rabbit polyclonal antibody, Proteintech, Rosemont, IL, USA (14732-1-AP)]; anti-TPD54 [1/1,000 dilution; rabbit polyclonal antibody, Proteintech (11795-1-AP)]; anti-hemagglutinin (HA) [1/1,000 dilution; rabbit polyclonal antibody, Clontech, Mountain View, CA, USA (631207)]; anti-green fluorescent protein (GFP) [1/1,000 dilution; rabbit polyclonal antibody, Santa Cruz, Dallas, TX, USA (sc-8334)]; or anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [1/10,000 dilution; mouse monoclonal antibody, Sigma-Aldrich (G9295)] antibody and horseradish-peroxidase-conjugated secondary antibody (for rabbit, donkey polyclonal antibody (NA934V); for mouse, sheep polyclonal antibody (NA931V) (GE Healthcare UK Ltd., Buckinghamshire, UK) as described previously (39). Subsequent washings were conducted using TBS-T. The protein bands were visualized using Amersham ECL Western Blotting Detection reagents (GE Healthcare UK Ltd.) and a ChemiDoc XRS Plus ImageLab System (Bio-Rad).

Clinical samples

All samples were acquired from patients who underwent treatment for squamous cell carcinoma in Showa University Dental Hospital from January 2001 through March 2015. All patients provided informed consent before enrollment in the study, in accordance with the protocol approved by the Institutional Review Board at Showa University Dental Hospital (approval no. DH2015-013). Primary lesions were resected from tongue (Fig. 2A-a-c and B-a, -b) and gingiva (Fig. 2B-c). Differentiation grade was determined according to the pathological reports submitted from the Division of Pathology, Department of Oral Diagnostic Sciences, School of Dentistry, Showa University.

Figure 2 Expression of TPD52, -53 and -54 in oral squamous carcinoma specimens. Representative resected squamous carcinoma tissues from the tongue (A-a-c and B-a, -b) and gingiva (B-c) were stained with H&E and were immunohistochemically stained for TPD52, -53 and -54, cytokeratin 10/13 [positive control (47)], and pre-immune IgG (negative control). Optical microscopic images were captured at low (×100) and high (×400, area enclosed by a black box in the ×100 image) magnification. Arrows indicate cancer pearl structures. Differentiation grade (high or poor) was determined according to pathological reports. Bars, 200 µm (×100) and 100 µm (×400).

Cell culture

SAS (40), HSC2, and HSC3 cells (41) (human oral squamous cell carcinoma-derived cell lines) were grown in high-glucose Dulbecco's modified Eagle's medium (HDMEM) (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin (Thermo Fisher Scientific) at 37°C in an atmosphere containing 5% CO2.

Molecular constructs, small interfering RNA (siRNA) and gene transfection

The coding regions of human tpd52 and -53 cDNAs were amplified by an RT-PCR technique using single-stranded cDNA reverse-transcribed from total RNA of SAS cells that was used as a template. All of the primer sequences used are shown in Table I. Final concentrations of the primers were 10 µM, respectively. PCR cycles were 95°C for 3 min; then, 94°C for 30 sec, 60°C for 30 sec, 72°C for 1 min (×40 cycles); and followed by 72°C for 5 min. The amplified products were subcloned into a pGEM T-Easy vector (Promega, Madison, WI, USA) using a TA-cloning technique. For construction of expression vectors of hemagglutinin (HA)-tagged human tpd52 and -53, cDNA of the coding region was amplified by PCR employing a sense-BglII-adaptor primer and an antisense KpnI-adaptor primer. The amplicons were double-digested with BglII and KpnI and inserted into the corresponding site of the pCMV-HA vector (Clontech Laboratories) as described previously (39). The resulting expression vectors were confirmed by sequencing using an ABI PRISM 310 Genetic Analyzer (Thermo Fisher Scientific) and the BigDye Terminator v3.1 Cycle Sequencing kit (Thermo Fisher Scientific). The expression vector for human tpd54 was that used in a previously described experiment (40). siRNA against human tpd52 and -54 was purchased from Sigma-Aldrich and Thermo Fisher Scientific, respectively. Control siRNA was purchased from Thermo Fisher Scientific. Expression vectors and siRNAs were transfected with Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's protocol.

Table I Primer sequences for molecular constructs.

Table I

Primer sequences for molecular constructs.

	Primer sequences
TPD52
Sense	5′-GTCTGCTTATCAGGAGGGGC-3′
Antisense	5′-GGCAGTGGGTAGCAGAACAA-3′
TPD53
Sense	5′-GAGGTAACCAGAAGCGGCTA-3′
Antisense	5′-ACAATGTCAAGGCCTGGGTT-3′
HA-tagged
TPD52
Sense	5′-GAAGATCTACATGGATTGTAGAGAGATGGA-3′
Antisense	5′-GGGGTACCTCACAGGCTCTCCTGTGTCTTT-3′
HA-tagged
TPD53
Sense	5′-GAAGATCTACATGGAGGCGCAGGCACAAGG-3′
Antisense	5′-GGGGTACCTTAGCACTGCAGCTCCTCCTCC-3′

[i] The sequences of primer pairs for cloning the coding regions of human tpd52, -53 and for constructing HA-tagged tpd52, -53 cDNAs are shown.

Generation of stable clones of cell lines

tpd52 and tpd54 overexpressing (OE) stable clones were obtained by using GFP Fusion TOPO TA Expression kits (Thermo Fisher Scientific). Single-stranded cDNAs were reverse-transcribed from total RNA of SAS cells that was used as a template. The amplified products were subcloned into a pcDNA3.1/NT-GFP-TOPO vector using TOPO cloning technology (Thermo Fisher Scientific). pcDNA3.1/NT-GFP (Thermo Fisher Scientific) was used as a control vector for OE experiments. TPD54 knocked-down (KD) stable clones were obtained by using BLOCK-iT Pol miR RNAi Expression Vector kits (Thermo Fisher Scientific). A double-stranded oligo targeting tpd54 was prepared by annealing two single-stranded oligos. The amplified products were subcloned into a pcDNA6.2-GW/Em-GFP-miR vector using Gateway cloning technology. pcDNA6.2-GW/Em-GFP-miR-neg (Thermo Fisher Scientific) was used as a negative control miRNA vector. All of the primer sequences used are shown in Table II. The resulting expression vectors were verified by sequencing and were transfected with Lipofectamine 2000 (Thermo Fisher Scientific). To obtain tpd52 and tpd54 OE stable clones, transfected SAS cells were cultivated in the presence of 0.5 mg/ml of Geneticin (Thermo Fisher Scientific). tpd54 knock-down stable clones were obtained by cultivation in the presence of 50 µg/ml of Blasticidin S HCl (Thermo Fisher Scientific), according to the manufacturer's protocol.

Table II Primer sequences for the generation of stable clones.

Table II

Primer sequences for the generation of stable clones.

	Primer sequences
TPD52 OE
Sense	5′-ATGGATTGTAGAGAGATGGA-3′
Antisense	5′-TCACAGGCTCTCCTGTGTCTTT-3′
TPD54 OE
Sense	5′-ATGGACTCCGCCGGCCAAGATATCAACCTG-3′
Antisense	5′-TTAGAAAGGTGCGGGATCCGACAGGGGCTT-3′
TPD54 KD
Sense	5′-TGCTGTTCAAATTCATGCAAACGCGGGTTTTGGCCACTGACTGACCCGCGTTTATGAATTTGAA-3′
Antisense	5′-CCTGTTCAAATTCATAAACGCGGGTCAGTCAGTGGCCAAAACCCGCGTTTGCATGAATTTGAAC-3′

[i] The sequences of primer pairs for TOPO cloning of tpd52 and −54 [TPD52 and −54 overexpressed (OE)], and oligo DNAs for generating miRNAs for tpd54 [TPD54 knockdown (KD)] are shown. The underlined sequences are the TPD54 targeting sequences.

Cell growth assay

One thousand cells were seeded into a 96-well tissue culture plate in triplicate. After 48 h, cell growth was assayed using the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as described previously (42). The experiment was performed in triplicate.

Anchorage-independent growth assay

The anchorage-independent growth assay was carried out by using a commercial kit (CytoSelect 96-Well In Vitro Tumor Sensitivity assay, Cell Biolabs, San Diego, CA, USA) according to the manufacturer's protocol. In total, 2500 transfected cells were grown in soft agar. After a week, the cells were examined under a microscope (Eclipse TS100/TS100-F, Nikon, Tokyo, Japan), photographed with a digital CCD camera (DS-Fil, Nikon), and formed colonies were counted (colonies/field). A colony was defined as a cell cluster that was >50 µm in diameter. Thereafter, cell growth was assayed using the MTT assay. The experiment was performed in triplicate.

Cell migration and invasion assays

Chemotaxis, haptotaxis, and invasion assays were carried out using a commercially available Boyden chamber kit (Chemotaxicell; Kurabo, Osaka, Japan). For the chemotaxis assay, the cells were starved in FBS-free medium for 24 h and then 10,000 of the transfected cells were seeded into a chamber inserted in a 24-well culture plate containing 10% FBS as a chemotactic agent. After 48 h, the cells were fixed with 10% formalin (Wako) and stained using crystal violet (43). Migrated cells were photographed, and the numbers of migrated cells were counted (cells/field). For the haptotaxis assay, the membranes were pre-coated with type I collagen (Nitta Gelatin Inc., Osaka, Japan), according to the manufacturer's protocol. Approximately 100,000 transfected cells were seeded into a chamber inserted in a 24-well culture plate. After 48 h, the cells were fixed and stained using crystal violet. Migrated cells were photographed, and were then counted, as described for the chemotaxis assay. The wound healing assay was carried out as described previously (44). The transfected cells (100,000) were seeded into a 24-well culture plate. Once the cells were confluent, a wound was made by scratching (0.9 mm width). After wound closure was first observed, the cells were fixed, stained using crystal violet and photographed. The wound width was then measured. For the invasion assay, the cells were starved in FBS-free medium for 24 h. Similar to the chemotaxis assay, 10,000 of the transfected cells were seeded into a chamber, which was pre-coated with type I collagen (Nitta Gelatin Inc.), and was then inserted in a 24-well culture plate. After 48 h, the cells were fixed and stained using crystal violet. Migrated cells were photographed and were counted in a manner similar to the chemotaxis and haptotaxis assays. Each experiment was performed in triplicate.

Mice

This study was approved by the Institutional Animal Care and Use Committee (approval no. 15024) and was carried out according to the Showa University Guidelines for Animal Experiments. Four-week-old female balb/c nu/nu mice (n=3 for each experimental group) were purchased from Clea Japan, Inc. (Tokyo, Japan) and were maintained under pathogen-free conditions. Approximately 1.0×107 cells in 100 µl of phosphate-buffered saline (PBS) were injected subcutaneously into a unilateral flank. Tumor-bearing mice were treated with 1 nmol of siRNAs (Koken, Tokyo, Japan) in 100 µl of AteloGene (Koken), which were injected into subcutaneous spaces around tumor sections once a week for 7 weeks from day 7. The AteloGene and siRNA mixture was generated according to the manufacturer's protocol. At the end of the experiment, mice were sacrificed by CO2 asphyxiation, and resected tumor, liver, and lung were fixed in formalin (Wako) and stained with hematoxylin and eosin (H&E) (Sakura Finetek Japan, Tokyo, Japan), as described previously (45). Tumor volume was determined by direct measurement, and was calculated using the formula π/6 × (large diameter) × (small diameter)2 (45). The Kaplan-Meier method was used for survival analysis (46).

Immunohistochemistry

Resected specimens were fixed with 10% formalin, embedded in paraffin, stained with H&E, and then immunohistochemically stained for TPD52 family proteins as described previously (46). Antigen retrieval was carried out with citrate-phosphate buffer (0.01 M, pH 6.0) at 121°C for 20 min. Endogenous peroxidases were blocked by incubating the sample with 10% H2O2 (Wako) for 10 min. Proteins were blocked using a commercial kit (Dako, Carpinteria, CA, USA) according to the manufacturer's protocol. The sections were incubated at 4°C overnight with primary antibodies (anti-TPD52 antibody, 1:50 dilution, rabbit polyclonal antibody; Biorbyt, Cambridgeshire, UK (orb100564); anti-TPD53 antibody, 1:200 dilution, rabbit polyclonal antibody, Proteintech (14732-1-AP); anti-TPD54 antibody, 1:200 dilution, rabbit polyclonal antibody, Proteintech (11795-1-AP); anti-cytokeratin 10/13, 1/200 dilution, mouse monoclonal antibody, Santa Cruz (DE-K13); control pre-immune IgG, 1/200 dilution, mouse IgG, Santa Cruz (sc-2762). The next day, the sections were incubated with secondary antibodies (EnVision+ system-HRP labelled polymer anti-rabbit/anti-mouse; Dako). Finally, sections were reacted with a 3, 3′-diaminobenzidine (DAB) peroxidase substrate kit (Dako) for color development and were examined under a microscope.

Statistical analysis

All values are expressed as means ± standard deviation of triplicate data sets. The statistical significance of differences between groups was analyzed using a paired Student's t-test. A p-value of <0.05 was considered statistically significant.

Compliance with ethical standards

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of Showa University at which the studies were conducted. Animal experiments were approved by the Institutional Animal Care and Use Committee and were carried out according to the Showa University Guidelines for Animal Experiments (approval no. 15024). Regarding the management of specimens, before enrollment in the study, informed consent was obtained from all individual participants included in the study, in accordance with the protocol approved by the Institutional Review Board at Showa University Dental Hospital (approval no. DH2015-013). The experiments complied with the current laws of Japan.

Results

TPD52 family proteins form hetero-complexes

Since previous studies (2,4) showed that TPD52 family proteins form homo-and hetero-complexes in various cells, we investigated whether such molecular interactions were observed in SAS cells by a co-immunoprecipitation (co-IP) assay (Fig. 1). In western blotting of the total cellular protein (input), the blotted bands of TPD54, TPD53, and TPD52 gave strong, weak, and faint signals, respectively, in agreement with our previous study (39). Of more importance, TPD52, -53, and -54 were found to co-immunoprecipitate with each other. Co-IP with control pre-immune mouse IgG indicated the specificity of the binding, despite the presence of a very faint pseudo-positive signal that was probably due to insufficient washing of the blots.

Figure 1 Co-immunoprecipitation analysis of TPD52 family proteins in SAS cells. Lysates of SAS cells were immunoprecipitated (IP) with anti-TPD52, -53, or -54 antibodies or with pre-immune mouse IgG. Subsequently, the elu-ates were analyzed by western blotting (WB) for co-precipitated TPD52, -53 and -54. GAPDH was blotted as a control. In the leftmost lane, 20 µg of total cellular proteins were loaded (input). At the left and right sides of the blot, the target proteins and co-precipitated IgG, together with their corresponding molecular weights, are shown.

TPD54 is highly expressed in OSCC

We recently showed that TPD54 is highly expressed in oral squamous cell carcinomas (38). However, the expression of TPD52 and -53 in OSCC, and the differences in TPD52 family protein expression in highly and poorly differentiated tumors are still unclear. We therefore analyzed the detailed expression patterns of TPD52 family proteins in OSCC using immunohistochemical staining (Fig. 2). Three cases each of highly and poorly differentiated tumors were randomly chosen from tongue (Fig. 2A-a-c and B-a, -b) and gingiva (Fig. 2B-c). Differentiation grade was determined according to the pathological reports. Immunohistochemical staining showed that cytokeratin 10/13 [a marker of SCC cells (47)] was highly expressed in all of these OSCCs. A cancer 'pearl' structure was observed in some of the highly differentiated tissues (arrows in Fig. 2 indicate a typical cancer pearl structure), but was not observed in poorly differentiated tissue. Additionally, staining with pre-immune IgG (negative control) indicated the specificity of TPD52 family immune staining. In agreement with our previous study (38), TPD54 was highly expressed in the cancer region and the surrounding connective tissue, regardless of the tumor differentiation level. In contrast, the expression of TPD52 in either highly or poorly differentiated OSCC was lower than that of TPD54, and TPD52 was barely expressed in normal tissue. TPD53 was moderately expressed in the cancer region.

TPD54 inhibited colony formation in an anchorage-independent manner

The higher expression level of TPD54 compared to TPD52 and -53 suggested that TPD54 might play a more important role in OSCCs. Furthermore, our previous study (39) showed that TPD54 affects cell attachment to the ECM and cell migration of OSCC cells. Based on those findings, we hypothesized that TPD54 might be a key protein in OSCC cells, and might negatively affect tumor progression mediated by TPD52 and -53. To further assess this possibility, tpd52, -53, and -54 were overexpressed in SAS cells. Additionally, in this experiment, HA-tagged tpd52 or -53 expression vectors were transfected into SAS cell lines that stably expressed GFP-tpd54 or GFP-miRNA against-tpd54. Subsequent western blot analysis (Fig. 3) showed that protein expression resulting from transient overexpression of tpd52 or -53 and stable overexpression or knock-down of tpd54 was observed as expected. Expression marker tags (HA and GFP) and internal control (GAPDH) proteins were also detected. Since expression profiles of control miRNA cell lines were similar to those of control cell lines (Fig. 3), protein expression in the control miRNA cell lines in further experiments are not shown to simplify the experiments. These cells were then assayed using an MTT assay of the monolayer cultured cells (Fig. 4A), a soft agar colony formation assay (Fig. 4B and C) and an MTT assay of the colonies formed in soft agar (Fig. 4D). Significant differences in cell growth between the control and several of the differently transfected cells were observed in the MTT assay of monolayer cultures (*p<0.05 versus control cells) (Fig. 4A), but the differences were smaller than those between the cells in colony formation assays (Fig. 4B–D). Colony formation of the tpd54-overexpressing cells was greatly limited in an anchorage-independent manner versus control cells, regardless of the co-expression of tpd52 or tpd53 (Fig. 4B). Conversely, knock-down of tpd54 enhanced the number of colonies formed (Fig. 4C). MTT assay of the cells in this colony forming experiment showed no differences in cell growth (Fig. 4D), indicating that cell viability was not an important factor even in an anchorage-dependent culture. These findings suggested that TPD54 might be capable of attenuating the tumorigenicity of other TPD52 family proteins, and that TPD54 and other TPD52 family proteins might have opposite effects on primary tumor formation. Next, it was examined whether these effects on cell growth are reproduced in other OSCC cell lines, or not. The same experiments were carried out in HSC2 (Fig. 5) and HSC3 (Fig. 6) cells. HSC2 and HSC3 cells also showed significant differences between transfected cells versus control cells in an anchorage-independent manner in colony formation (*p<0.05 versus control cells) as well as in cell growth in a monolayer culture. However, the effects observed in HSC2 and HSC3 cells were slightly different than those observed in SAS cells. Therefore, SAS cells, in which OE and KD of TPD52 proteins were the most effective were used as representative OSCC cells in the following experiments.

Figure 3 Western blot analysis of SAS cells with overexpression or knock-down of tpd54 and/or overexpression of tpd52 or -53. Control-HA and HA-tpd52 and -53 expression vectors were transfected into cells of the SAS cell line in which GFP-empty (control), GFP-tpd54 (TPD54 OE) genes, GFP-tpd54miRNA (TPD54 KD), or control miRNA were stably expressed, or not (untransfected). After 48 h, total cellular proteins were collected and analyzed by western blotting for TPD52, -53, and -54, HA, GFP, and GAPDH (an internal control) expression.

Figure 4 Effects of overexpression or knock-down of tpd54 and/or of overexpression of tpd52 and -53 on cell proliferation in SAS cells. Control-HA (−), HA-tpd52 (TPD52 OE), or -53 (TPD53 OE) expression vectors were transfected into SAS cells, in which GFP-empty (control) or GFP-tpd54 (TPD54 OE) genes or GFP-tpd54miRNA (TPD54 KD) were expressed. Cell growth was then analyzed using an MTT assay of cells in either monolayer culture (A) or in soft agar culture (D). Phase contrast analysis of colony formation in soft agar (B and C), and quantification of colony number are also shown. (A) MTT assay of monolayer cultures. The value in control cells was designated as '1' and relative values are shown. *p<0.05 versus control. (B-D) Colony formation and MTT assay of soft agar cultures. Bar, 200 µm (B). The total number of colonies (black bars) and the number of colonies with a diameter >100 µm (white bars) were counted under a microscope. *p<0.05 versus control (C). At the end of the colony forming assay, the cells were analyzed using an MTT assay. The value in control cells was designated as '1' and relative values are shown (D).

Figure 5 Effects of overexpression or knock-down of tpd54 and/or of overexpression of tpd52 and -53 on cell proliferation in HSC2 cells. Control-HA (−), HA-tpd52 (TPD52 OE), or -53 (TPD53 OE) expression vectors were transfected into HSC2 cells, in which GFP-empty (control) or GFP-tpd54 (TPD54 OE) genes or GFP-tpd54miRNA (TPD54 KD) were expressed. Cell growth was then analyzed using an MTT assay of cells in either monolayer culture (A) or in soft agar culture (D). Phase contrast analysis of colony formation in soft agar (B and C), and quantification of colony number are also shown. (A) MTT assay of monolayer cultures. The value in control cells was designated as '1' and relative values are shown. *p<0.05 versus control. (B-D) Colony formation and MTT assay of soft agar cultures. Bar, 200 µm. (B) The total number of colonies (black bars) and the number of colonies with a diameter >100 µm (white bars) were counted under a microscope. *p<0.05 versus control (C). At the end of the colony forming assay, the cells were analyzed using an MTT assay. The value in control cells was designated as '1' and relative values are shown (D).

Figure 6 Effects of overexpression or knock-down of tpd54 and/or of overexpression of tpd52 and -53 on cell proliferation in HSC3 cells. Control-HA (−), HA-tpd52 (TPD52 OE), or -53 (TPD53 OE) expression vectors were transfected into HSC3 cells, in which GFP-empty (control) or GFP-tpd54 (TPD54 OE) genes or GFP-tpd54miRNA (TPD54 KD) were expressed. Cell growth was then analyzed using an MTT assay of cells in either monolayer culture (A) or in soft agar culture (D). Phase contrast analysis of colony formation in soft agar (B and C), and quantification of colony number are also shown. (A) MTT assay of monolayer cultures. The value in control cells was designated as 1, and relative values are shown. *p<0.05 versus control. (B-D) Colony formation and MTT assay of soft agar cultures. Bar, 200 µm (B). The total number of colonies (black bars) and the number of colonies with a diameter >100 µm (white bars) were counted under a microscope. *p<0.05 versus control (C). At the end of the colony forming assay, the cells were analyzed using an MTT assay. The value in control cells was designated as '1' and relative values are shown (D).

TPD54 downregulates the cell migration of OSCC-derived cells

Our previous report (39) showed that TPD54 plays a crucial role in the cell migration and invasion of OSCC cells. To further assess the effects of TPD52, -53, and -54 on the cell migration of OSCC cells, we performed chemotaxis, haptotaxis and wound healing assays of the transfected cells (Fig. 7). tpd54 overexpression reduced cell migration versus control cells in a chemotaxis assay when expressed either alone or in combination with overexpression of tpd52 or -53 (Fig. 7A). However, little effects on haptotaxis were observed following overexpression or knock-down of tpd54 alone or combined with overexpression of tpd52 or -53 (Fig. 7B). Overexpression of tpd52 or -53 alone did not affect either chemotaxis or haptotaxis. tpd54-overexpressing cells (tpd54+, 53−, 52−) showed decreased wound closure compared to control in a wound healing assay (*p<0.05, Fig. 7C). No differences versus control were observed for the other experimental groups (Fig. 7C). We additionally investigated the effect of the different cell transfection combinations on cell invasion (Fig. 7D). However, cell invasion was barely modulated by any of these combinations. These results indicated that TPD54 might regulate cell migration through the modulation of molecular events such as integrin signaling.

Figure 7 Effects of overexpression or knocking-down of tpd54, and/or overexpression of tpd52 and -53 on cell migration and invasion. Control-HA (−), or HA-tpd52 (TPD52 OE), -53 (TPD53 OE) expression vectors were transfected into SAS cells in which GFP-empty (control), GFP-tpd54 (TPD54 OE) genes, or GFP-tpd54shRNA (TPD54 KD) was stably expressed. One hundred thousand transfected cells were analyzed in chemotaxis (A), haptotaxis (B), wound healing (C), and cell invasion (D) assays. (A) Chemotaxis assay. Left and right panels show optical microscopic images and a graph of the number of migrated cells, respectively. Bar, 500 µm. *p<0.05 versus control. (B) Haptotaxis assay. Left and right panels show optical microscopic images and a graph of the number of migrated cells, respectively. Bar, 100 µm. (C) Wound healing assay. Left and right panels show optical microscopic images and a graph of wound widths, respectively. Bar, 100 µm. *p<0.05 versus control. (D) Cell invasion assay. Left and right panels show optical microscopic images and a graph of the number of migrated cells, respectively. Bar, 200 µm. *p<0.05 versus control. Details of each assay can be found in Materials and methods.

tpd54 overexpression attenuated tumor growth in vivo

Since our previous in vitro study suggested that TPD54 has negative effects on cell proliferation and migration in OSCC cells, we further examined the ability of TPD54 to modulate tumor growth and/or metastasis in an in vivo study. The scheme of this study is outlined in Fig. 8A. In these experiments, one nanomole of siRNAs in Atelogene was injected once a week into each mouse (n=3 per group) with a xenografted tumor of OSCC cells, in order to obtain sustainable knock-down of tpd52 or -54. A significant difference in body weight between the tpd54 overexpressing group and control was seen (*p<0.05) (Fig. 8B) although no significant tumor suppression was observed in the tpd54 overexpressing group (Fig. 8C and E). However, contrary to our expectations, knock-down of tpd52 in tpd54 overexpressing cells showed little effect on tumor growth. No significant differences in survival rate between the different groups of mice, as calculated by Kaplan-Meier's method, were observed (Fig. 8D). At the endpoint, specimens of the original tumor, and of lung and liver tissue were H&E stained for confirmation of metastasis (Fig. 9). However, there were few histological differences between the groups. Metastasis was not observed in any group by microscopic examination. These results indicated that TPD54 might function as a negative regulator of tumor growth, suggesting that interactions between TPD52 family protein members might be involved in the growth of OSCC.

Figure 8 Effects of overexpression or knock-down of tpd52 and -54 on body weight, tumor growth and survival rate in tumor-xenografted mice. SAS cells, in which GFP-empty (control), GFP-tpd54 (TPD54 OE), GFP-tpd54miRNA (TPD54 KD), GFP-tpd52 (TPD52 OE), GFP-tpd54 with tpd52siRNA (TPD54 OE with TPD52si), or GFP-tpd52 with tpd54siRNA (TPD52 OE with TPD54si) stably expressed were used. (A) A schematic outline of the tumor-xenograft study. (B) The average body weight of the mice. (C) The average tumor volume of the mice. (D) Kaplan Meier analysis of the survival rate of the mice. (E) Representative photographic images of tumor-xenografted mice at the end point (day 49).

Figure 9 Histopathological evaluation of the original tumor, and of lung and liver from tumor-xenografted mice. SAS cells, in which GFP-empty (control), GFP-tpd54 (TPD54 OE), GFP-tpd54miRNA (TPD54 KD), GFP-tpd52 (TPD52 OE), GFP-tpd54 with tpd52siRNA (TPD54 OE with TPD52si) or GFP-tpd52 with tpd54siRNA (TPD52 OE with TPD54si) stably expressed were used. All slides were photographed at low (×40; bar, 1 mm) and high (×200; bar, 200 µm) magnification.

Discussion

Tumor protein D52 (TPD52) family members are expressed in several types of cancers, including OSCC (5–17). Additionally, the cellular functions of this family have been widely studied. Previous reports indicated that overexpression of tpd52 in nonmalignant 3T3 fibroblasts induced an increase in the growth rate and also enabled the cells to exist in an anchorage-independent manner in vitro and to undergo metastatic growth in vivo (18). Moreover, overexpression of tpd52 led to increased tumor growth in cancer cells (48). Downregulation of tpd52 led to a decrease in cell migration, invasion (23), and apoptosis (20). TPD53 regulates the cell cycle and is highly upregulated at the G2-M phase transition (25). However, little is known about the cellular functions of TPD54, particularly in OSCCs. We recently showed that TPD54 is expressed in OSCCs and in nearby hypertrophic squamous cells, and we suggested that this protein might be a candidate marker of oral epithelial carcinogenesis (38). The expression of TPD52 and -53 in OSCC and differences in their expression in high and poor differentiation stages of OSCC are still unclear. We therefore determined the detailed expression patterns of TPD52 family proteins in OSCC (Fig. 1). Indeed, TPD54 was highly expressed in the cancer region, regardless of the differentiation stage. However, TPD52 was expressed at a lower level than TPD54 in either highly or poorly differentiated OSCC. TPD53 was moderately expressed in the cancer region. A previous report (4) indicated homo- or heteromeric interactions between TPD52, -53, and -54 and suggested that these proteins might play several kinds of roles in cancer progression. These combined data suggested that TPD52 and TPD54 interact with each other and that these proteins, in particular TPD54, play important roles in cancer progression in OSCC. In the present study, co-IP experiments (Fig. 1) confirmed interactions between those TPD family proteins, suggesting that they do form hetero-protein complexes on OSCC cells. Furthermore, in our previous experiments involving tpd54 overexpression or knock-down in OSCC-derived SAS cells, we observed little effects on cell proliferation, caspase activity, MMP activity or cell invasion in monolayer cultures (39). However, tpd54 overexpression led to a decrease in anchorage-independent proliferation, regardless of apoptosis (39). In the present study, we investigated in more detail the cellular effects of expression levels of TPD52 family proteins by using gene co-expression. These studies indicated little differences in cell proliferation between several differently transfected cells in a monolayer culture. However, in anchorage-independent culture, strong suppression of colony formation was observed in tpd54 overexpressing cells, regardless of the co-expression of tpd52 or tpd53. In addition, knock-down of tpd54 enhanced colony formation. The correlation between anchorage-independent proliferation and the in vivo behavior of cells is well established (49,50). The combination of these data with our previous results suggested a significant involvement of TPD54 in tumor growth in the modulation of cancer metastasis. These results prompted us to further assess the combinatorial effects of TPD52 family proteins on cell migration and invasion. These assays showed that TPD54 negatively controls cell migration, leading us to hypothesize that TPD52 proteins might modulate the affinity of integrins for the ECM (39).

Since these in vitro results suggested that TPD54 has negative effects on cell proliferation and migration in OSCC cells, we examined the involvement of TPD54 in tumor growth in vivo. In those experiments, a significant difference in body weight between tpd54 overexpressing mice and controls was seen, although no significant tumor suppressive effect was observed in this group. However, tumor growth in several other experimental groups with different combinations of TPD-family proteins did not agree with our expectations. We therefore consider that several as yet unknown molecular pathways might be involved, and/or that homo- and heteromeric interactions of TPD52 family proteins through their coiled-coil motif with each other or with other heteromeric partners (4,26,28,29,51–53) are likely to participate in tumor growth regulation by these proteins. Li et al (54) reported that prostate leucine zipper (PrLZ), which is a TPD52 isoform, is highly expressed in prostate cancers and interacts with the androgen receptor. We consider that this interaction may also occur in OSCC cells. Analysis of the effects of such an interaction on consequent interactions of TPD52 family proteins might be essential in order to solve this issue. The details of TPD52 family interactions are still unclear, particularly in OSCC cells. We are currently in the process of investigating the cellular growth, invasion and metastasis mediated by the interaction of TPD52 family protein members. He et al recently reported that TPD54 has a role in the promotion of cancer progression in OSCC cell lines (55), which is the opposite result to the present study. This discrepancy may be due to the different cells used in the two studies. Thus, the CAL27 and KB cell lines that they employed in their experiments are an oral adenosquamous carcinoma cell line (56) and a HeLa contaminant-epidermal carcinoma cell line (57), respectively. However, the OSCC cells that we employed are SAS cells, which were isolated from tongue OSCC (40). These cells might harbor cells with more of the original phenotypes of OSCC cells. In any case, more detailed investigations are required in the future in order to address this discrepancy.

For tumor therapy, molecular targeted therapies are currently in the spotlight, next to surgery, radiation, and chemotherapy. TPD52 family proteins are also considered as novel candidate target proteins since these proteins are expressed in many types of cancers (31). In conclusion, our study showed that TPD54 acts as a negative regulator of anchorage-independent proliferation and cell migration of OSCC cells in vitro. Moreover, TPD54 decreased body weight gain and tended to attenuate tumor growth in vivo. These combined data suggest that an increase in the expression of tpd54 might improve outcomes in OSCC patients. As yet, only a few studies of TPD54 have been reported. Further investigation of TPD54 is still required.

Acknowledgments

This study was supported by Grants-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (JSPS) [KAKENHI C to Y.M. (15K11301), S.K. (24593060) and T.S. (15K11269)]. The whole manuscript was proofread by FOLTE (Tokyo, Japan). The authors wish to thank all of the staff at the Department of Oral and Maxillofacial Surgery, School of Dentistry, Showa University for their helpful suggestions; Dr Kenji Mishima of the Division of Pathology, Department of Oral Diagnostic Sciences, School of Dentistry, Showa University for pathological diagnostic advice; and Ms. Miho Yoshihara for secretarial assistance.

References