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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2006-0155
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 6 2373-2379
Copyright © 2006 by The Endocrine Society

Ribonucleic Acid Interference Targeting S100A4 (Mts1) Suppresses Tumor Growth and Metastasis of Anaplastic Thyroid Carcinoma in a Mouse Model

Yufei Shi, Minjing Zou, Katharine Collison, Essa Y. Baitei, Zaha Al-Makhalafi, Nadir R. Farid and Futwan A. Al-Mohanna

Departments of Genetics (Y.S., M.Z., E.Y.B.) and Biological and Medical Research (K.C., Z.A.-M., F.A.A.-M.), King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia; and Osancor Biotech Inc. (N.R.F.), Watford, Herts WD17 3BY, United Kingdom

Address all correspondence and requests for reprints to: Yufei Shi, MBC 3, Department of Genetics, King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Saudi Arabia. E-mail: yufei{at}kfshrc.edu.sa.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: The characteristic feature of malignant neoplasm is invasion and metastasis. Despite advances in the management of thyroid carcinoma and other solid tumors, metastasis continues to be the most significant cause in cancer mortality.

Objective: Our objective was to examine the effects of S100A4 expression knockdown by RNA interference on the growth and metastasis of human anaplastic thyroid carcinoma cells (ARO) and the sensibility of ARO to paclitaxel after S100A4 knockdown.

Design: A plasmid construct was made that expressed small hairpin RNA (shRNA) specific for S100A4. The construct was stably transfected into ARO cells (ARO/S100A4-shRNA). The tumorigenicity, metastatic potential, and sensibility of ARO/S100A4-shRNA to paclitaxel were investigated.

Results: S100A4 expression was reduced by 71.3 ± 4.7% in ARO/S100A4-shRNA by real-time RT-PCR analysis. The growth rate of ARO/S100A4-shRNA was reduced by 46 ± 7.6% in a cell proliferation assay. Cell cycle analysis showed increased G2/M accumulation in ARO/S100A4-shRNA. Tumor formation and growth induced by sc injection of 5 x 106 ARO/S100A4-shRNA into the nude mice were significantly reduced, and no tumor metastasis was found in any of the mice. We also demonstrated significant induction of apoptosis in ARO/S100A4-shRNA after incubation with 15 nM paclitaxel, indicating that tumor cells were sensitized to chemotherapy as a result of S100A4 knockdown.

Conclusions: These data suggest that reduction of S100A4 by RNA interference is a viable approach to inhibit tumor growth and metastasis. Given that S100A4 is overexpressed in many kinds of tumors, the current study provides the proof of concept in its therapeutic potential.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE S100A4 HAS RECENTLY emerged as an important protein with the capacity to promote invasion and metastasis of many human neoplasms (1, 2, 3, 4, 5, 6). S100A4 is a member of the S100 calcium-binding proteins that regulate intracellular processes such as cell growth, motility, cell cycle, transcription, and differentiation (7). Twenty members of the S100 protein family have been identified so far, and altogether, S100 proteins represent the largest subgroup in the EF-hand Ca2+-binding protein family (7). S100 proteins have received increasing attention because of their close association with several human diseases such as cardiomyopathy (8), neurodegenerative disorders (9), and cancer (2). These proteins have also been shown to be valuable markers in the diagnosis and management of these diseases and are considered to have a potential as drug targets to improve therapies (10, 11, 12, 13).

Thyroid carcinomas of follicular cell origin are the most common endocrine malignancies (14). Although the survival rate of patients with well-differentiated thyroid carcinoma exceeds the rate for most other cancers, the development of metastasis continues to be the most significant cause in thyroid carcinoma mortality (14, 15). Therefore, identification of genes involved in this process will enable us to target them for future diagnosis or therapeutic intervention.

We have previously demonstrated that S100A4 is highly expressed in an anaplastic thyroid carcinoma cell line (ARO) with high metastatic potential and found that S100A4 overexpression is associated with advanced disease stage and metastasis (16, 17). To further investigate its role in thyroid tumor metastasis and its potential as a therapeutic target, in the present study, we introduced into ARO cells a plasmid vector expressing a small hairpin RNA (shRNA) to inhibit S100A4. The effects of reduced S100A4 gene expression by RNA interference on tumor growth and metastasis were examined. The results suggest that S100A4 gene knockdown could significantly suppress anaplastic thyroid carcinoma cell growth and metastasis in a mouse model.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Female BALB/c ({nu}/{nu}) nude mice 6–8 wk of age were used as model hosts for anaplastic thyroid tumors. To establish sc thyroid tumor, mice were injected sc with 5 x 106 ARO cells into the lower flank. Palpable tumors developed within 10–14 d. Tumor volume was calculated by the following formula: width2x length x 0.5. Mice were killed for pulmonary metastasis 60 d after tumor cell inoculation.

Cell line and cell culture

ARO, a human anaplastic thyroid carcinoma cell, was propagated in DMEM containing 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37 C in a humidified atmosphere containing 5% CO2. The human S100A4 hairpin oligo targeting S100A4 exon 2 (5'-GATCCGGGTGACAAGTTCAAGCTCTTCAAGAGAGAGCTTGAACTTGTCACCCTCTTTTTTGGAAA-3') was designed by selecting appropriate sequences from the human S100A4 mRNA. The oligo was annealed with its complementary strand. The double-stranded shRNA oligo was then cloned into pSilencer 3.1-H1 vector (pS100A4-shRNA) according to the manufacturer’s instructions (Ambion Inc., Austin, TX). The construct was verified by DNA sequencing. As a control, the green fluorescent protein (GFP) hairpin oligo was also cloned into pSilencer 3.1-H1 vector (pGFP-shRNA).

Expression of pS100A4-shRNA in ARO cells

ARO cells were transfected with either pS100A4-shRNA or control pGFP-shRNA as described previously (18). Cells were selected in the presence of 200 µg/ml hygromycin for 3 wk. The resulting stable clones (ARO/S100A4-shRNA or ARO/GFP-shRNA) were pooled and used for in vitro and in vivo studies. In a subset of experiments, the cells were incubated with 15 nM paclitaxel for 24 h and then subjected to flow cytometry analysis for apoptosis.

Quantitative real-time RT-PCR analysis for S100A4

Total RNAs from ARO/S100A4-shRNA and ARO/GFP-shRNA were extracted by guanidinium thiocyanate-phenol-chloroform method. Two micrograms of total RNA were reverse transcribed using the Promega RT system (Promega, Madison, WI). The LightCycler DNA Master SYBR Green kit was used for quantitative real-time PCR analysis as described previously (16). The resulting concentration of S100A4 PCR products was normalized by comparison with glyceraldehyde-3-phosphate dehydrogenase and was used to determine the S100A4 mRNA level in the ARO cells.

Confocal microscopy

Both ARO/S100A4-shRNA and ARO/GFP-shRNA cells grown in coverslips were fixed in 3.7% (vol/vol) formaldehyde. S100A4 was visualized by indirect immunofluorescence using polyclonal rabbit anti-S100A4 antibody (Dako, Carpinteria, CA) and tetramethylrhodamine isothiocyanate-conjugated goat antirabbit secondary antibody (Pierce, Rockford, IL). The S100A4 expression was analyzed using a Leica TCS True confocal microscope (Heidelberg, Germany).

Cell proliferation and viability assay

Both ARO/S100A4-shRNA and ARO/GFP-shRNA cells were seeded in 96-well plates with a concentration of 1 x 104 cells per well in triplicate. Cell proliferation was determined after 24 h by bromodeoxyuridine (BrdU) incorporation assay using Promega’s BrdU cell proliferation assay kit. For cell viability assay, 1 x 104 cells per well in triplicate were treated with different concentrations of paclitaxel for 24 h at 37 C. Cell viability was determined using Promega’s CellTiter-Glo luminescent cell viability assay kit.

In vitro tumor invasion assay

The tumor invasion assay was performed to quantitate the relative degree of invasiveness of ARO, ARO/S100A4-shRNA, and ARO/GFP-shRNA carcinoma cells, as described previously (19).

Flow cytometry analysis for cell cycle and apoptosis

For cell cycle analysis, 1 x 105 nonadherent cells were fixed in 70% ethanol for 20 min. The cell pellets were washed in cold PBS and incubated for 30 min in PBS containing 10 µg/ml propidium iodide and 1 µg/ml RNase A at 37 C. Propidium iodide fluorescence was quantified using the FL-2 channel. Fragmented apoptotic nuclei were recognized by their subdiploid (sub-G1) DNA content. For apoptosis analysis, 1 x 105 cells were double stained with fluorescein isothiocyanate-conjugated annexin V and propidium iodide for 15 min at room temperature (apoptosis detection kit; Roche, Mannheim, Germany) and then analyzed on a FACScan flow cytometer (BD Biosciences, San Jose, CA). Annexin V and propidium iodide emissions were detected in the FL-1 and FL-2 channels, respectively.

Statistical analyses

Tumor volume among different groups was analyzed by Mann-Whitney U nonparametric test to identify groups having significantly different tumor sizes. Student’s t test was used to evaluate statistical significance. Differences were considered statistically significant when the P value was <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Knockdown of S100A4 expression by RNA interference in ARO cells

shRNA cDNA constructs for human S100A4 and GFP were stably expressed in ARO cells after plasmid transfection and antibiotic selection. As shown in Fig. 1AGo, quantitative real-time RT-PCR analysis of S100A4 mRNA from S100A4-shRNA and GFP-shRNA transfected ARO cells as well as untransfected ARO cells showed 71.3 ± 4.7% of reduction of S100A4 mRNA in ARO/S100A4-shRNA cells compared with ARO/GFP-shRNA cells (P < 0.01). There was no significant difference in S100A4 knockdown between ARO/GFP-shRNA cells and untransfected ARO cells. The real-time RT-PCR results were further confirmed by confocal microscopy analysis of S100A4 protein in ARO/S100A4-shRNA and ARO/GFP-shRNA cells, which showed a 3-fold reduction in S100A4 protein expression in ARO/S100A4-shRNA (Fig. 1BGo).


Figure 1
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  		FIG. 1. Knockdown of S100A4 gene expression in human anaplastic thyroid carcinoma cell line ARO by S100A4-shRNA. shRNA cDNA constructs for GFP and human S100A4 were stably expressed in ARO cells after plasmid transfection and antibiotic selection. A, Real-time RT-PCR analysis of S100A4 mRNA in S100A4-shRNA and GFP-shRNA transfected ARO cells as well as untransfected ARO cells; B, confocal microscopy analysis of S100A4 protein in S100A4-shRNA vs. GFP-shRNA transfected ARO cells: a, ARO cells stained with goat antirabbit secondary antibody alone (background control); b, ARO/GFP-shRNA cells stained with S100A4 and goat antirabbit secondary antibody; c, ARO/S100A4-shRNA cells stained with S100A4 and goat antirabbit secondary antibody.

 
Reduction of cell proliferation and tumor growth after S100A4 knockdown

The effect of S100A4 protein reduction on ARO cell proliferation was examined. Cell proliferation was measured by BrdU uptake after S100A4 knockdown in ARO cells. As shown in Fig. 2AGo, cell proliferation in ARO/S100A4-shRNA cells was reduced by 46 ± 7.6% compared with that in ARO/GFP-shRNA cells. We also examined the ability of ARO cells to produce tumors in nude mice after S100A4 knockdown. For this purpose, groups of 10 nude mice each were injected sc with 5 x 106 ARO/S100A4-shRNA cells or ARO/GFP-shRNA cells (control group). As shown in Fig. 2BGo, there was a 1- to 2-wk delay in tumor formation, and the tumor growth rate in nude mice was significantly reduced after S100A4 knockdown. Four of 10 mice did not develop tumor, whereas all of the 10 control mice developed tumor. The tumor load measured 60 d after inoculation was 2 ± 0.3 g in the mice receiving ARO/GFP-shRNA cells vs. 0.5 ± 0.2 g in the mice receiving ARO/S100A4-shRNA cells (Fig. 2CGo) (P < 0.01).


Figure 2
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  		FIG. 2. Reduction of cell proliferation in vitro and tumor growth in vivo by S100A4 knockdown in ARO cells. A, BrdU uptake in ARO/S100A4-shRNA cells compared with that in ARO/GFP-shRNA cells after 24 h incubation at 37 C. Data are presented as mean ± SEM of three experiments. B, Reduction of tumor growth rate in nude mice (n = 10) after sc injection of 5 x 106 ARO/S100A4-shRNA cells. Four of 10 mice did not develop tumor, whereas all the 10 mice developed tumor after sc injection of 5 x 106 ARO/GFP-shRNA cells. Data are presented as mean ± SEM of tumor volume. C, Effects of S100A4 knockdown on tumor burden. Two groups of nude mice (10 in each group) were inoculated sc with 5 x 106 ARO/S100A4-shRNA and ARO/S100A4-shRNA cells, respectively. Sixty days after inoculation, the tumors were removed and weighed. Data are presented as mean ± SEM of tumor weight.

 
Reduction of invasive potential after S100A4 knockdown

The in vitro tumor invasion assay was performed to quantitate the relative degree of invasiveness of ARO/S100A4-shRNA, ARO/GFP-shRNA, and ARO cells through a transwell insert coated with basement membrane Matrigel. As shown in Fig. 3Go, there was no significant difference in the invasiveness between ARO and ARO/GFP-shRNA (92.2 ± 3.9% for ARO cells vs. 100% for ARO/GFP-shRNA). However, there was a significant reduction of invasiveness for ARO/S100A4-shRNA cells (7.7 ± 1.5%) compared with that of ARO/GFP-shRNA cells (P < 0.01). No tumor metastasis was seen 90 d after sc injection of 5 x 106 ARO/S100A4-shRNA cells, whereas pulmonary metastasis foci could be found in mice 50 d after receiving 5 x 106 ARO/GFP-shRNA cells (data not shown).


Figure 3
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  		FIG. 3. Effect of S100A4 knockdown on the invasive potential of ARO carcinoma cells. An in vitro invasion assay was carried out to compare and quantify the invasiveness of ARO, ARO/S100A4-shRNA, and ARO/GFP-shRNA cells. The cells were labeled with 1 µCi/ml 125I-deoxyuridine and placed in the upper compartment of a Transwell chamber for 96 h. The tumor cells penetrating into the lower surface of Matrigel-coated filters were recovered with trysin-EDTA and counted in a gamma counter. The experiment was performed in triplicate and repeated three times. Data were expressed as a mean ± SEM of three experiments.

 
Enhancement of paclitaxel-induced apoptosis after S100A4 knockdown

Paclitaxel is an anticancer agent and inhibits cell cycle progression by accumulating cells in M phase. We suspected that the growth suppression of ARO cells by the RNA interference-mediated knockdown of S100A4 was caused by disruption of cell cycle transition with delay in mitotic entry and were interested in knowing whether paclitaxel could enhance the effect. Therefore, the effect of S100A4 knockdown on cell cycle and apoptosis before and after paclitaxel treatment was examined. To determine whether the growth suppression of ARO cells by S100A4 knockdown was caused by disruption of cell cycle transition, we analyzed the DNA contents of ARO/S100A4-shRNA and ARO/GFP-shRNA cells. As shown in Fig. 4AGo, an increase in the G2/M accumulation in the cell cycle was found in ARO/S100A4-shRNA cells. Treatment with 15 nM paclitaxel for 24 h caused even more G2/M accumulation in both ARO/S100A4-shRNA and ARO/GFP-shRNA cells and increase in the sub-G1 populations (apoptotic cells) in ARO/S100A4-shRNA cells. We next analyzed apoptosis of ARO/S100A4-shRNA and ARO/GFP-shRNA cells by annexin V and PI staining. As shown in Fig. 4BGo, a higher level of spontaneous apoptosis (2-fold) was found in ARO/S100A4-shRNA cells and an additional 2-fold increase after 15 nM paclitaxel treatment compared with ARO/GFP-shRNA cells (P < 0.01) (Fig. 4BGo). These results indicate that the S100A4 knockdown not only led ARO cells to the increased accumulation in the G2/M phase and eventual apoptosis but also enhanced the cytotoxicity of paclitaxel.


Figure 4
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  		FIG. 4. Effect of S100A4 knockdown on the cell cycle and apoptosis before and after paclitaxel treatment. A, ARO/S100A4-shRNA and ARO/GFP-shRNA cells were incubated with 15 nM paclitaxel for 24 h and then fixed with ethanol and stained with propidium iodide. The histograms represent the emission detected at the FL-2 channel by flow cytometry. There is increased G2/M accumulation in ARO/S100A4-shRNA cells before treatment, and most cells arrested in G2/M phase after treatment in both ARO/S100A4-shRNA and ARO/GFP-shRNA cells. A profound sub-G1 area in ARO/S100A4-shRNA cells after treatment represents the apoptotic changes. The experiments were repeated three times, and representative results are shown. B, Annexin V and propidium iodide (PI) (detected in the FL-1 and FL-2 channels, respectively) staining of ARO/S100A4-shRNA and ARO/GFP-shRNA cells. A higher level of spontaneous apoptosis was observed in ARO/S100A4-shRNA, and a 2-fold increase in apoptosis was found in ARO/S100A4-shRNA cells after 15 nM paclitaxel treatment for 24 h. Cells mostly display annexin V-positive and propidium iodide-negative staining, suggestive of apoptosis. The experiments were repeated three times, and representative results are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
S100A4 is known to be involved in tumor invasion and metastasis by virtue of its ability to activate nonmuscle myosin (20, 21, 22, 23, 24). Numerous clinical studies showed a correlation between high S100A4 expression and a poor prognostic outcome in a variety of human cancers such as breast, ovary, colon, gastric, thyroid, and pancreatic carcinomas (6, 16). In an attempt to investigate the possibility that S100A4 might be a therapeutic target, we employed the RNA interference technique for knockdown of its expression in the anaplastic thyroid carcinoma cell line ARO and its phenotype. We found that stable knockdown of S100A4 strikingly inhibited cell proliferation and invasiveness in vitro and tumorigenicity and metastasis in a mouse xenograft model. The knockdown induced accumulation of cells in the G2/M phase and eventual apoptosis. We further showed that the knockdown of S100A4 significantly enhanced the cytotoxic effect of paclitaxel, a commonly used chemotherapy drug.

The involvement of S100A4 in tumor metastasis has been well documented in the rodent model systems (25, 26, 27, 28). In a recent study, Grum-Schwensen et al. (29) have demonstrated a significant delay in tumor development in S100A4 knockout mice after injection of highly metastatic mouse mammary carcinoma cells, CSML100. Moreover, tumors that developed in these mice did not metastasize. Coinjection of CSML100 cells with immortalized S100A4-secreting fibroblasts partially restored the dynamics of tumor development and metastasis. Their results indicate an important role of host-derived stroma cells expressing S100A4 in tumor growth and metastasis (29). Our results suggest that suppression of tumor growth and metastasis can also be achieved by knockdown of S100A4 in thyroid cancer cells. We found four of the 10 mice did not develop tumor after injection of S100A4 knockdown ARO cells, whereas tumors occurred in all of the 10 control mice. Because we used pooled S100A4 knockdown clones in the study, the variation of S100A4 expression in different cells in their population may impact on the frequency of tumor occurrence and the delay in tumor occurrence. It would be reasonable to predict that the cell population with the least small interfering RNA (siRNA) or S100A4 knockdown could contribute preferentially to early tumor onset and faster tumor growth. An earlier study showed reversal of the in vivo metastatic phenotype of human osteosarcoma cells by an anti-S100A4 ribozyme (30). However, in vitro and in vivo cell proliferation and tumorigenicity were unchanged, which is contradictory to our current results. The differences may relate to characteristics of individual cell lines.

Paclitaxel is an anticancer agent from the taxane class of drugs. It binds to free tubulin and promotes the assembly of tubulin into stable microtubules by interfering with their disassembly. It inhibits cell cycle progression by accumulating cells in M phase at the metaphase-anaphase transition and subsequently leads them to apoptosis. Knockdown of S100A4 also induced accumulation of cells in the G2/M phase and an increase in apoptosis. Our results suggest that the combination of S100A4 knockdown and paclitaxel could result in strong impairment of M phase progression and the synergistic induction of apoptosis. The mechanism that triggers apoptosis by S100A4 knockdown remains to be clarified. Taxanes have cytotoxic activity against many types of cancers including thyroid cancer. Accumulation of G2/M phase and subsequent induction of apoptosis in anaplastic thyroid cancer cells by low doses of Taxol (6–50 nM) has been reported recently (31). These taxane-mediated chemotherapies could be more effective in combination with knockdown of S100A4.

Hata et al. (32) have recently reported interesting findings in pancreatic cancer cell lines that suppression of tumor growth, accumulation of cells in the G2/M phase and eventual apoptosis, and synergistic enhancement of the cytotoxicity of taxanes can be achieved by RNA interference-mediated knockdown of AURKA, the gene encoding Aurora A kinase. Aurora A kinase is involved in the regulation of centrosomes and segregation of chromosomes and is frequently amplified and overexpressed in various kinds of human cancers, including thyroid cancer, which overexpressed all three Aurora kinases (A, B, and C) (33). Knockdown of both S100A4 and Aurora kinases by RNA interference may have a synergistic effect on the reduction of thyroid tumor growth. Indeed, Sorrentino et al. (34) have demonstrated significant growth inhibition of thyroid anaplastic carcinoma cells by blocking Aurora B expression through RNA interference.

RNA interference is becoming a common application for in vivo cancer therapy (35, 36). Although S100A4 is ubiquitously expressed, the level of expression is much higher in thyroid tumor tissues and their metastatic foci, making it a good candidate for gene silencing (16, 17). Local injection of S100A4-shRNA into tumor through plasmid-, adenovirus-, or lentivirus-mediated RNA interference is feasible and may be beneficial to control tumor growth and metastasis, particularly in combination with paclitaxel. A new gene transfer method using a biomaterial, atelocollagen, prepared from bovine dermis has been developed (37). Takei et al. (38) have shown that the vascular endothelial growth factor siRNA with atelocollagen dramatically suppressed tumor angiogenesis and tumor growth in a PC-3 sc xenograft model. Atelocollagen is unique in that it is a liquid at 4 C and a gel at 37 C. Therefore, atelocollagen can increase cellular uptake, nuclease resistance, and prolonged release of siRNA administered into the tumor. Effective systemic delivery of S100A4-shRNA to metastatic foci is challenging because of safety concerns for virus-based vectors and effectiveness of plasmid or naked DNA vectors (degradation of the naked DNA by nucleases can be a problem). However, Spänkuch et al. (39) have recently demonstrated a significant antitumor effect by iv administration of aurintricarboxylic acid-treated plasmid DNA carrying shRNA against Polo-like kinase 1. Aurintricarboxylic acid is a nuclease inhibitor used as protection against nucleases in blood. A similar approach might be adopted to treat metastatic foci using plasmid DNA carrying S100A4-shRNA.

In summary, we have investigated the effect of S100A4 knockdown on thyroid cancer cell growth, metastasis, and chemosensitivity in a mouse xenograft model. Our findings indicate that S100A4 is an attractive candidate for a therapeutic target, because it can regress tumorigenicity and metastasis and enhance chemosensitivity to paclitaxel. The current study provides the proof of concept in its therapeutic potential and warrants further development.


   Footnotes
 
All of the authors have nothing to declare.

First Published Online March 21, 2006

Abbreviations: BrdU, Bromodeoxyuridine; GFP, green fluorescent protein; shRNA, small hairpin RNA; siRNA, small interfering RNA.

Received January 24, 2006.

Accepted March 14, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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