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Keywords:

  • THY1;
  • nasopharyngeal carcinoma;
  • tumor suppressor gene;
  • antiinvasive;
  • tetracycline-regulated gene expression

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

THY1 was previously identified as a candidate tumor suppressor gene (TSG) associated with lymph node metastases in nasopharyngeal carcinoma (NPC) through functional studies. It was identified by oligonucleotide microarray analysis as an interesting differentially expressed gene. However, direct functional evidence is still lacking for THY1 being a TSG in NPC, as in vivo tumorigenicity assays have not been previously reported in our last study of THY1. In this study, a tetracycline-inducible expression vector, pETE-Bsd, was used to obtain stable transfectants of THY1. The stringent in vivo tumorigenicity assay results show that the activation of THY1 suppresses tumor formation of HONE1 cells in nude mice, and the tumor formation ability was restored in the presence of doxycycline (a tetracycline analog), when the gene is shut off. Functional inactivation of this gene is observed in all the tumors derived from the tumorigenic transfectant. The tumor suppressive effect could be repressed by knockdown of THY1 expression in nontumorigenic microcell hybrids. Further studies indicate that expression of THY1 inhibits HONE1 cell growth in vitro by arresting cells in G0/G1 phase. It greatly reduces the ability for anchorage-independent growth. The invasiveness of HONE1 cells was also inhibited by the expression of THY1. These findings suggest that THY1 is a TSG in NPC, which is involved in invasion and shows an association with tumor metastasis. Taken together, THY1 clearly plays an important functional role in tumor suppression in NPC.

THY1 (CD90) is a cell surface glycoprotein of 25–37 kDa localized on the outer leaflet of cell membranes, enriched in lipid raft microdomains. Human THY1 maps to chromosome 11q22.3. It is expressed on various cell types, including human fibroblasts, neurons, blood stem cells and endothelial cells as well as murine T cells.1 THY1 is involved in T cell activation and the other nonimmunologic functions including inhibition of neurite outgrowth, apoptotic signaling, leukocyte and melanoma cell adhesion and migration and fibroblast proliferation and migration.2, 3 Taken together, these functions suggest that THY1 is an important mediator of cell–cell and cell–matrix interactions. In T cells, it is reported to function in cell adhesion with the bone marrow stroma.1 Surface expression of THY1 promotes focal adhesion in fibroblasts.2 In cancers, THY1 was initially found to be differentially expressed in tumorigenic and nontumorigenic hybrid clones derived from the transfer of chromosome 11 into the human ovarian cancer cell line SKOV-3.4 The later functional studies also suggest that THY1 is associated with tumor suppression in human ovarian cancer.5 A recent study shows that loss of THY1 expression is correlated with poor survival rate in neuroblastoma patients.6 THY1 is also involved in the adhesion of melanoma cells to the human dermal microvascular endothelial cells.7 THY1 is a cell surface marker, which has been used to identify local and circulating liver cancer stem cells.8, 9

Nasopharyngeal carcinoma (NPC) is a malignancy that is common in Southern China among the ethnic Chinese. Epstein-Barr virus, genetic predisposition, dietary and environmental factors are all believed to be involved in NPC development.10–12 Losses or inactivation of tumor suppressor genes (TSGs) are important steps in tumor development. Using oligonucleotide microarray analysis, THY1, which maps close to a previously defined 11q22-23 NPC critical region,13, 14 is identified as a candidate TSG in NPC. THY1 is exclusively expressed in the nontumorigenic chromosome 11 microcell hybrids (MCHs) and is downregulated in the tumor revertants, NPC cell lines and primary tumor tissues. The mechanism responsible for THY1 gene inactivation in these cell lines is attributed to hypermethylation. Downregulated THY1 protein expression is significantly associated with lymph node metastases. However, direct functional evidence is still lacking for THY1 being a TSG in NPC, as in vivo tumorigenicity assays have not been previously reported.15 In this study, we focus on the functional role of THY1 in the tumorigenesis of NPC. Using an inducible gene expression vector,16THY1 stable transfectants were obtained for this study, and in vivo tumorigenicity assay for THY1 transfectants was performed. To further strengthen the tumor suppressor role of THY1 in NPC, the gene knockdown of THY1 in a nontumorigenic HONE1/chromosome 11 MCH was performed to study its impact on tumorigenicity. Stably transfected tumor suppressive THY1 cell lines were further examined by in vitro growth assays including cell cycle analysis and the soft agar assay. As reported in our previous study, by using a tissue microarray and immunohistochemical staining, we show that the frequency of THY1-downregulated expression in lymph node metastatic NPC is significantly higher than in the primary tumor, suggesting that THY1 is significantly associated with lymph node metastasis.15In vitro cell invasion and migration assays were performed to examine the antimetastasis potential of THY1 in NPC in this study.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cell lines and culture conditions

The recipient NPC HONE1-2 cell line and the HONE1/chromosome 11 MCH cell line, HK11.19, were maintained, as previously described.15 This chromosome 11 MCH contained an extra intact chromosome 11 transferred to the recipient HONE1 cell. In a previous study,13 we described how this MCH cell line was subsequently injected into nude mice and exhibited a delayed latency period before tumor formation. The immortalized nasopharyngeal epithelial cell lines, NP69 and NP460, were cultured, as described.17, 18 Construction of a pETE-Bsd responsive vector and a HONE1 cell line, HONE1-2, producing the tetracycline transactivator tTA, was described in Protopopov et al.16 Stable THY1 and vector-alone transfectants were maintained, as described.19 The THY1 knockdown clones were maintained in culture medium containing 500 μg/ml neomycin and 100 μg/ml zeocin.

Reverse transcription-polymerase chain reaction and quantitative real-time RT-PCR

Cell lines were cultured until 80% confluent, and their total RNAs were subsequently extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA). Total RNAs from the cell lines were extracted and converted to first-strand cDNA using M-MLV Reverse Transcriptase (USB, Cleveland, OH). The primer sequences of THY1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used, and PCR was performed as previously described.15, 19

Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed using THY1-specific primer pair and probe (Applied Biosystems, Foster City, CA).19 Human GAPDH primer and probe reagents (Applied Biosystems) were used as the normalization control in subsequent quantitative analysis. Real-time RT-PCR was performed in a Step-One Plus machine using TaqMan PCR core reagent kits (Applied Biosystems) as described.20

Western blot analysis

Western blot analysis of THY1 was performed as previously reported.15 In brief, viable NPC cells at 70–80% confluency (5 × 106) were harvested and then lysed. Samples of 10 μg cellular protein were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (pore size: 0.45 μm; Millipore, Billerica, MA). The membranes were incubated with primary antibodies H-110 (Santa Cruz Biotechnology, Santa Cruz, CA) for the THY1 protein and Ab-1 (Calbiochem, Darmstadt, Germany) for α-tubulin. The signals were visualized by enhanced chemiluminescence method according to the manufacturer's instructions (Amersham, Uppsala, Sweden).

Transfection and gene expression analysis

The full-length THY1 cDNA was inserted into pETE-Bsd plasmid, and stable THY1-expressing clones were obtained by using Lipofectamine 2000 (Invitrogen) as described.15

Knockdown of THY1 analysis in HK11.19 cells

The THY1 RNA knockdown was achieved by using the BLOCK-iT Inducible H1 RNAi Entry Vector Kit system (Invitrogen, Carlsbad, CA). In brief, 3 pairs of THY1 shRNA oligonucleotides were designed by BLOCK-iT RNAi designer program (https://rnaidesigner.invitrogen.com/rnaiexpress). The sequences of the 3 pairs of the THY1 shRNA oligonucleotides are as follows: 5′-CACCGCTCTCCTGCTAACAGTCTTGCGAACAAGACTGTTAGCAGGAGAGC-3′ and 5′-AAAAGCTCTCCTGCTAACAGTCTTGTTCGCAAGACTGTTAGCAGGAGAGC-3′, 5′-CACCGAACCAACTTCACCAGCAAATCGAAATTTGCTGGTGAAGTTGGTTC-3′ and 5′-AAAAGAACCAACTTCACCAGCAAATTTCGATTTGCTGGTGAAGTTGGTTC-3′ and 5′-CACCGCAAATACAACATGAAGGTCCCGAAGGACCTTCATGTTGTATTTGC-3′ and 5′-AAAAGCAAATACAACATGAAGGTCCTTCGGGACCTTCATGTTGTATTTGC-3′, which target at nucleotide positions, 78–99, 298–117 and 301–321, respectively, of the human THY1 cDNA (NM_006288). The shRNA oligonucleotides were ligated into a linearized pENTR/H1/TO plasmid. The shRNA plasmids were transiently transfected into the recipient cell line 11.19, and total RNA was extracted and RT-PCR was performed.

In vivo tumorigenicity assay

The tumorigenicity of cell lines was assayed, as reported by Lung et al.19 In brief, 1 × 107 cells were injected into 4- to 8-week-old female athymic Balb/c Nu/Nu mice. A total of 6 sites were tested for each cell line, and tumor growth in animals was checked weekly. If tumor formation was observed, tumors were subsequently excised from the sites of injection, and a representative tumor was reconstituted into cell culture for subsequent molecular analyses. For inhibition of the tetracycline-inducible expression of THY1 in vivo, 200 μg/ml dox was added to the drinking water of mice 1 week before injection, and the water with doxycycline (dox, a tetracycline analog) was changed twice a week.

In vitro cell growth analysis

The cell growth of the THY1 transfectants and the vector-alone clones ±0.2 μg/ml dox was assayed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. In brief, 2 × 103 cells were seeded on Day 0, and cell growth was measured every other day until Day 5. A volume of 30 μl of MTT (Sigma Chemical, St. Louis, MO) solution (5 mg/ml) was then added and cells were further incubated at 37°C for 2 h. The OD at 540 nm was determined with a Opsys MR microplate reader (Thermo Labsystems, Chantlly, VA).

FACS analysis

The cell cycle distribution of THY1-C2 and -C9 and Bsd-C1 ±0.2 μg/ml dox were analyzed by flow cytometry, as previously described by Lung et al.19 In brief, 1 × 106 cells were seeded and allowed to attach to the bottom of a T25 culture flask overnight. The cells were collected and fixed with cold 70% ethanol and were then treated with 1 U of DNase-free RNase and incubated for 30 min at 37°C. A total of 1 mg/ml propidium iodide (Sigma Chemical, St. Louis, MO) was added directly to the cell suspension, and a total of 10,000 fixed cells were analyzed by FACScan (Becton Dickinson, San Jose, CA).

Soft agar assay

The soft agar assay was performed as described by Cheung et al.20 A quantity of 5 × 104 cells were mixed with 2 ml of agar ±0.2 μg/ml dox (0.4% in DMEM), overlaid on 2 ml of 1% plating agar in DMEM and seeded on a 6-well culture plate. After 10 days, the cells were stained with neutral red and colonies were counted.

Cell invasion and migration assays

The cell invasion and migration assays were performed as described by Leung et al.21 In brief, a quantity of 2 × 105 cells ±0.2 μg/ml dox were seeded into the chamber of a 24-well micropore membrane filter with 8-μm pores (Becton Dickinson Labware, Franklin Lakes, NJ) for migration assay or seeded into the chamber of a 24-well Matrigel-coated membrane filter (Becton Dickinson Labware, Franklin Lakes, NJ) for invasion assay. The bottom chamber was filled with DMEM ±0.2 μg/ml dox containing 10% FBS as a chemoattractant. After 36-hr incubation at 37°C, membranes were fixed and stained with crystal violet. All of the cells invading through the membrane were counted under an inverted light microscope at 10× magnification.

Statistical analysis

The 1-way ANOVA test was used to calculate whether the tumor growth kinetics between the various THY1 transfected/knockdown cell lines and the vector-alone control was significantly different in the in vivo tumorigenicity assays. Student's t-test was used to determine the confidence limits in group comparisons for the in vitro assays. For both ANOVA and Student's t-tests, a p value ≤ 0.05 was regarded as statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Activation of THY1 expression in HONE1 cells suppresses tumor formation in vivo

As shown in Figures 1a and 1b, the gene and protein expression levels of THY1 in transfected clones, THY1-C2, -C9 and -C10, were induced in the absence of dox and analyzed by quantitative real-time PCR and Western blot analyses, respectively. Dox treatment inactivates the tTA transcriptional activator, resulting in reduction of transgene and protein expression. Reduction of THY1 transcript and protein levels was observed following dox treatment in THY1 stable clones. By Western blotting, a high expression was detected with THY1-C2 (−dox); the THY1 levels only dropped to those observed in the vector-alone control with the THY1-C9 (+dox).

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Figure 1. (a) Real-time PCR analysis of THY1 gene expression in pETE-Bsd transfectants THY1-C2, -C9, -C10 and Bsd-C1 (±dox). The HONE1-2 cell line, producing the tetracycline transactivator tTA, was used to obtain stable transfectants. Relative expression (ddCt) of THY1 in each cell line compared with Bsd-C1 was studied. (b) Western blot analysis of THY1 in the same THY1 transfectants and Bsd-C1 control (±dox). Alpha-tubulin was used as a loading control.

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After injection into nude mice, the vector-alone transfectant Bsd-C1 formed palpable tumors in all 6 injection sites within 2 to 3 weeks (Table 1). Tumorigenicity was suppressed by overexpressing THY1 (−dox), as observed with clones THY1-C2 and -C9 (Fig. 2a). No tumors appeared for THY1-C2 and only 2 tumors were observed with THY1-C9. The latency period of these 2 tumors derived from THY1-C9 was 5 to 6 weeks, and it is significantly slower than that of the vector-alone clone (Table 1). This is in contrast to the appearance of tumors in all 6 sites for THY1-C9 (+dox), when the THY1 expression level is reduced. The average tumor size of THY1-C9 (+dox) was 569 mm3, whereas when the gene was turned on, the average size was reduced to 151 mm3, 5 weeks after injection; the difference was statistically significant (p value = 0.037). Even though the THY1 expression was suppressed in THY1-C9 (+dox) and its growth rates were slower than that of the vector-alone clone Bsd-C1, the difference is not statistically significant (Table 1, Fig. 2a). THY1-C2 (+dox) showed a clear growth inhibition effect. This may be attributed to the higher levels of THY1 expressed because of leakiness in the system, as detected by the high levels of THY1 expression by RT-PCR and Western blot analyses (Figs. 1a and 1b).

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Figure 2. (a) The tumor growth curves for vector-alone and THY1 stable transfectants (±dox) represent an average tumor volume of all sites inoculated for each cell population. (b) Loss of expression of THY1 in tumors and tumor segregants derived from tumorigenic transfectants. RT-PCR analysis of THY1 in tumors THY1-C10-T1 to -T6 and its representative tumor segregant, THY1-C10-T6-TS. Gene expression of THY1-C10 and Bsd-C1 served as positive and negative controls, respectively.

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Table 1. Tumorigenicity assays of THY1 transfectants and THY1-knockdown MCHs
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Loss of expression of THY1 in tumors and a tumor segregant derived from tumorigenic transfectants is associated with tumorigenicity

No significant tumor suppression was observed for the other THY1 stable transfectant, THY1-C10 (−dox), when compared with the vector-alone Bsd-C1 (−dox), as all 6 injection sites formed tumors (Table 1). The growth kinetics of THY1-C10 (−dox) was quite similar to that of THY1-C9 (+dox). The tumor growth kinetics for this THY1 transfectant is shown in Figure 2a. To check the status of THY1 following tumor formation in this tumorigenic THY1 clone, total RNA was isolated from each tumor, and cells from 1 tumor (T6) were reconstituted in culture medium. Results of RT-PCR analysis for tumors and the tumor segregant derived from THY1-C10 show that the THY1 gene expression was significantly reduced in tumors THY1-C10-T1 to -T5, and the reduced levels are comparable to THY1-C10 (+dox); loss of THY1 expression was observed in THY1-C10-T6 and its tumor segregant, THY1-C10-T6-TS (Fig. 2b). Because of this functional inactivation of THY1 during the inoculation in nude mice, THY1-C10 is not tumor suppressive. Therefore, this clone was not used for further functional studies.

Tumorigenicity is restored in THY1 knockdown in microcell hybrid cell lines

To further confirm the tumor suppressive effect of THY1 in NPC, a THY1 knockdown in the nontumorigenic HONE1/chromosome 11 MCH, HK11.19, which expresses high level of THY1 protein and gene expression,15 was carried out to observe its effect on tumor formation ability. The real-time RT-PCR results show that the gene expression of THY1 was induced in HK11.19 when compared with HONE1 cells (Fig. 3a). Stable THY1 knockdown transfectants were obtained by transfecting the shRNAs 78 and 301 into HK11.19. Reduction of THY1 gene expression was observed in both stable clones of each THY1 shRNA oligonucleotide. The 2 knockdown clones of shRNA301 show the greatest suppression of the THY1 expression (Fig. 3a), and they were chosen for the Western blot analysis and the subsequent tumorigenicity assay.

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Figure 3. (a) Real-time RT-PCR analysis of gene expression of THY1 in the stable THY1-knockdown MCHs. HK11.19 cells were stably transfected with the THY1 shRNA oligonucleotides, THY1-shRNA78 and THY1-shRNA301. HONE1 cells served as controls. (b) Western blot analysis of protein expression of THY1 in the stable THY1-knockdown MCHs, 11.19-THY1-shRNA301-C2 and -C4 and vector-alone transfectant. Alpha-tubulin was used as a loading control. (c) Tumor growth kinetics of THY1-knockdown HK11.19 clones, vector-alone pENTR-C1 and HONE1 cells. The average tumor volume of all sites inoculated for each cell population is presented.

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The protein expression of THY1 was significantly suppressed in the clones 11.19-THY1-301-C2 and -C4, and their protein expression levels were comparable to the HONE1 (Fig. 3b). Tumorigenicity was suppressed in the vector-alone clone 11.19-pENTR-C1, and tumors were not observed at any site of injection up to 10 weeks after inoculation (Table 1 and Fig. 3c). Tumors appeared for both THY1-knockdown clones in at least 4 injection sites, which showed similar growth kinetics; the differences were statistically significant when compared with the vector-alone control (p values = 0.003 and 0.006, respectively). When compared with the tumor growth rate of HONE1 cells, those 2 THY1-knockdown MCHs were significantly slower (Table 1 and Fig. 3c).

THY1 inhibits HONE1 cell growth via G0/G1 phase arrest

Figure 4a shows that the growth rates of THY1-C2 and -C9, which express THY1 (−dox), were consistently slower than the vector-alone Bsd-C1, from days 1 to 5, and the inhibition is slightly higher in THY1-C2 from days 3 to 5. When THY1 expression was suppressed, the growth kinetics of THY1-C2 and -C9 (+dox) and Bsd-C1 (+dox) was very similar at all time points.

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Figure 4. (a) MTT analysis of cell growth of THY1-C2- and -C9 and Bsd-C1 transfectants (±dox) for 5 days. Data represent the OD detected in the 2 THY1 and vector-alone transfectants (±dox). “*” designates a statistically significant difference from the vector-alone transfectant (±dox) (p < 0.05). (b) FACS analysis of THY1-C2- and -C9 and Bsd-C1 (±dox). The average percentage of cells in G0/G1, S and G2/M phases is calculated from 3 independent experiments. Representative propidium iodide staining results of THY1-C2- and -C9 and Bsd-C1 (±dox) are shown. “*” designates a statistically significant difference from the vector-alone clone (p < 0.05), and “**” designates a statistically significant difference from the transfectant (+dox) (p < 0.05).

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To further study the growth inhibition mechanism of THY1, the cell cycle status of THY1-C2 and -C9 and Bsd-C1 was determined by DNA flow cytometry analysis. When THY1 was expressed in THY1-C2 and -C9 (−dox), there were significant increases in relative numbers of cells in G0/G1 phase from 46.3 to 55.4% and 55% and a significant decrease of cells in S phase from 27 to 15.9% and 16.1%, respectively (Fig. 4b). When THY1 expression is suppressed, the THY1-C2 and -C9 (+dox) cell cycle status is similar to that of Bsd-C1; the addition of dox to THY1-C2 and -C9 significantly increased the relative number of cells in S phase and reduced the number of cells in G0/G1 phase. Taken together with the MTT assay results, THY1 expression is associated with G0/G1 growth arrest.

THY1 reduces anchorage-independent growth of HONE1 cells

The transforming ability of HONE1 cells was tested in anchorage-independent soft agar assays performed with both suppressive THY1 transfectants and Bsd-C1 vector-alone control (Figs. 5a and 5b). The vector-alone clone (±dox) formed large colonies in soft agar, whereas both THY1-C2 and -C9 (−dox) rarely form colonies larger than 100 μm in diameter. For THY1-C9 (−dox), small colonies were visible in the soft agar. Very few colonies appeared in the other stable clone THY1-C2, when THY1 was expressed, and most remained as single cells. The numbers of large colonies were significantly increased, and the number of total colonies increased to numbers comparable to the vector-alone for THY1-C9 (+dox). With THY1-C2 (+dox), the numbers of small colonies appearing were significantly increased, but that of large colonies were not significantly affected. Hence, the soft agar assay results suggest that THY1 gene expression in HONE1 cells suppresses its colony formation ability in vitro in both sizes and numbers of colonies.

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Figure 5. (a) Anchorage-independent soft agar assay was performed with THY1-C2- and -C9 and Bsd-C1 transfectant cell lines (±dox). Representative results are shown. (b) The number of colonies formed was counted in each cell line. “*” designates a statistically significant difference from the vector-alone clone (p < 0.05), and “**” designates a statistically significant difference from the transfectant (+dox) (p < 0.05). (c) Transwell invasion assay of THY1 stable transfectants. The transwell filter was used to measure the invasion ability of THY1-C2- and -C9 and the Bsd-C1 control (±dox). Invading cells at the lower surface of the transwell filter were stained and counted. Representative results are shown. (d) The number of invading cells was counted in each cell line. “*” designates a statistically significant difference from the vector-alone clone (p < 0.05), and “**” designates a statistically significant difference from the transfectant (+dox) (p < 0.05).

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Overexpression of THY1 suppresses invasiveness of HONE1 cells

The antimetastasis potential of THY1 was examined by studying the invasiveness and mobility of NPC cells overexpressing THY1 in the in vitro invasion assay. The cell invasion and mobility assays were performed for the 2 THY1 stable transfectants and the vector-alone clone to see how many cells passed through the Matrigel-coated membrane or the membrane-alone. Figure 5c shows the microscopic views of the stained cells on the chamber membrane. When THY1 is overexpressed, the numbers of invasive cells were significantly reduced by 2.9- and 9.4-fold, respectively, in THY1-C2 and -C9 (−dox), when compared with Bsd-C1 (Fig. 5d), whereas with THY1-C2 and -C9 (+dox), numbers of invading cells increased. This effect is more obvious in THY1-C9, and the increase was statistically significant. For Bsd-C1 (±dox), there was no significant change in the number of invading cells. On the other hand, the migration potential of the 2 THY1 stable clones was not significantly affected when incubated under the same conditions as in the invasion assay (data not shown). Hence, the reduction of number of cells in the 2 THY1 clones observed in the invasion assay was not due to their change of migration ability.

Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This study provides the first functional evidence that THY1 expression is associated with both tumor suppression and invasion. Using a tetracycline-regulated vector system to control for clonal variation, in vivo tumorigenicity assays clearly show that the overexpression of THY1 is sufficient to induce potent tumor suppression in a nude mouse assay. Importantly, by knockdown of THY1 expression in a nontumorigenic MCH, we show that the THY1-knockdown clones revert to the tumorigenic phenotype of the parental HONE1-2 cells.

Furthermore, overexpression of THY1 in HONE1-2 cells results in reduced invasiveness. In our previous tissue microarray analysis,15 the frequency of THY1 downregulated expression in lymph node metastatic NPC was significantly higher than in primary tumors. Taken together with the present in vitro invasion assay results, loss of THY1 expression is correlated with enhanced invasiveness of NPC cells. In addition, the anchorage-independent growth of HONE1 was also suppressed by THY1. Similarly, oncogenic transformation of NIH3T3 cells by ras oncoproteins, resulting in anchorage-independent growth and soft agar colony formation, is associated with loss of THY1 surface expression.22 Results of the current in vitro soft agar, cell growth and cell cycle assays suggest that inhibition of G1 to S phase transition and anchorage-independent growth contribute to the in vivo tumor suppressive effects of THY1.

In this study, we show that both THY1-C2 and -C9 functionally suppress tumor formation, when the transgene is overexpressed in the absence of dox. However, only partial inhibition of tumorigenicity is observed for THY1-C9 and THY1-C2 still shows clear growth inhibition effects in vivo in the presence of dox. This may be explained by some leakiness in THY1 expression, as shown in both RT-PCR and Western blot analyses, as the addition of dox in vitro could not reduce the ectopic THY1 expression of both transfectants to the basal expression of HONE1 cells or the vector-alone clone. Moreover, it is likely that leakage is stronger in vivo than in vitro, as can be seen in this study and noted in previous studies using this inducible expression system.23–25 For the other tumorigenic transfectant, THY1-C10, tumors formed in all nude mice tested (−dox). The subsequent RT-PCR analysis showed that after inoculation of this clone in nude mice, both reduction and loss of mRNA expression of THY1 were observed in all 6 tumors and the representative tumor segregant derived from this clone. The reduced levels of THY1 expression in the 5 tumors (THY1-C10-T1 to -T5) are similar to the original clone when the THY1 expression is suppressed (+dox), which may explain the less tumorigenic properties of THY1-C10 when compared with the vector-alone clone. For THY1-C10-T6 and its tumor segregant, their THY1 gene expression is completely lost; indeed the tumor size of THY1-C10-T6 is largest (936 mm3) among all the 6 tumors derived from this THY1 stable clone. It is likely that loss of THY1 expression is associated with large tumor formation in NPC. This kind of functional inactivation due to elimination of the transgene overexpression has been observed in our previous study of the TSG Cell Adhesion Molecule 1 (CADM1, formerly called TSLC1) in NPC.19 Thus, it is clear from this functional inactivation that the loss of THY1 expression enables tumor growth of HONE1 cells in nude mice.

Although tumorigenicity results show that THY1-C2 (−dox) was more tumor suppressive in mice than THY1-C9 (−dox), results of the invasion assay show that THY1-C9 was likely to be more antiinvasive. The migration assay shows that the migration rate of THY1-C2 was slightly higher than that of THY1-C9 (−dox), although the change is not statistically significant (data not shown). This may explain why there were more invaded cells observed in THY1-C2, despite the higher expression level of THY1 expression in this clone.

Importantly, functional studies were also performed for the selected THY1-knockdown stable HK11.19 clones. Both reverted back to their tumorigenic phenotype, although the tumor growth kinetics was still lower than that of the original recipient HONE1 cells. These results indicate that although THY1 is critically involved in tumor suppression in HONE1 cells, other growth inhibitory gene(s) besides THY1 might be still present in chromosome 11. Our previous studies indicate that there is more than 1 critical region in chromosome 11 in NPC13, 14 and CADM1 and Alpha B-Crystallin, at the nearby 11q23.2-23.3 region, have been identified as a TSG and a putative TSG, respectively, in NPC in our previous studies.19, 26 It has been reported that in ovarian cancer THY1 might function as a tumor suppressor by upregulating FN and the antiangiogenic molecule TSP1.27 In contrast, our preliminary results in NPC show that the gene expression of TSP1 was not induced by both stable and transient THY1 transfection (unpublished observation). Instead, we showed that THY1 mediates its tumor suppressive function via the upregulation of CADM1 expression in NPC (unpublished observation). Although the reason for these disparate results is not known, it may indicate cell- or tissue-specific responses.

Using the tetracycline-regulated and shRNA knockdown systems in the current studies, we now clearly show that the activation of THY1 suppresses tumor formation in nude mice and this effect could be repressed by knockdown of THY1 expression. We now provide compelling functional evidence associating THY1 with tumorigenicity and invasiveness. As reported in our previous THY1 study,15 we showed that the downregulated THY1 expression is significantly associated with the lymph node metastatic NPC. Thus, THY1 is a TSG with antimetastasis activity in NPC.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors acknowledge the financial support from the Research Grants Council of the Hong Kong Special Administrative Region, People's Republic of China to M.L.L. and the Swedish Cancer Society, the Swedish Research Council, the Swedish Foundation for International Cooperation in Research and Higher Education (STINT), the Swedish Institute, the Royal Swedish Academy of Sciences, INTAS and Karolinska Institute to E.R.Z. The authors thank Dr. Eugene Hung (Hong Kong University of Science and Technology) for the gift of pENTR/H1/TO plasmid.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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