1{alpha},25-Dihydroxyvitamin D3 Targets PTEN-Dependent Fibronectin Expression to Restore Thyroid Cancer Cell Adhesiveness

Wei Liu, Sylvia L. Asa and Shereen Ezzat

Department of Pathology (W.L., S.L.A.), University Health Network and Toronto Medical Laboratories, Department of Medicine (S.E.), Mount Sinai Hospital, and Freeman Centre for Endocrine Oncology (S.L.A., S.E.), University of Toronto, Toronto, Ontario, Canada M5G 2M9

Address all correspondence and requests for reprints to: Dr. Sylvia L. Asa, Department of Pathology, 610 University Avenue, Suite 4-302, Toronto, Ontario, Canada M5G 2M9. E-mail: sylvia.asa{at}uhn.on.ca.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have previously reported that the hormonal form of 1{alpha},25-dihydroxyvitamin D3 (1,25-VD3), and its noncalciomimetic analog EB1089, arrest the growth of human thyroid cancer cells by increasing the cell cycle inhibitor p27. In the present study, we investigated whether the tumor-suppressive effects of vitamin D (VD) compounds may also be mediated by mechanisms that govern cell adhesiveness. Both 1,25-VD3 and EB1089 increased cell adhesiveness, an effect that was accompanied by consistent increases in fibronectin (FN) expression. Introduction of small interfering RNA against FN resulted in down-regulation of FN expression and diminished cell adhesiveness to a collagen-type I matrix. To determine whether this action of 1,25-VD3 was mediated through the PTEN/phosphoinositol 3-kinase pathway, we examined whether this tumor suppressor protein/dual phosphatase can influence FN expression and consequently cell adhesiveness Overexpression of wild-type PTEN induced FN expression as well as cell adhesiveness. In contrast, introduction of mutant forms of PTEN failed to induce FN and led to diminished cell adhesiveness. Conversely, small interfering RNA-mediated PTEN down-regulation attenuated FN expression as well as cell adhesiveness. The attenuated FN expression was also associated with relative insensitivity to 1,25-VD3 growth-suppressive action. Cells down-regulated for FN demonstrated a more aggressive growth pattern in xenografted mice and were also relatively insensitive to 1,25-VD3 treatment. Taken together, our findings highlight the significance of FN in modulating thyroid cancer cell adhesiveness and, at least in part, in mediating VD actions on neoplastic cell growth.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THYROID CARCINOMAS have a very wide spectrum of differentiation from some of the most indolent carcinomas (papillary microcarcinoma) to the most invasive and lethal human malignancies (anaplastic thyroid carcinoma) (1). This spectrum of progression has been closely linked with a pattern of cumulative genetic defects that correlate with tumor differentiation, metastatic potential, and aggressiveness (2, 3). Thus, thyroid cancer provides an ideal model in which to examine the effects of targeted modulation of cell growth and differentiation in human neoplasms of varying degrees of differentiation and with specific genetic defects.

In addition to its established role in the regulation of calcium homeostasis, the hormonal form of 1{alpha},25-dihydroxyvitamin D3 (1,25-VD3; also known as calcitriol), has been recognized to play a role in the modulation of proliferation and differentiation of several cell types (4, 5, 6, 7). Vitamin D (VD) has been reported to induce apoptosis in human breast carcinoma (8) and leukemic cells (9). Several VD analogs that are relatively free of the undesirable effects of hypercalcemia, hypercalciuria, and soft tissue calcification have been similarly demonstrated to exert antiproliferative effects, rendering them promising therapeutic tools (7). The actions of VD are thought to be mediated by the VD receptor (VDR), a transcription factor and member of the steroid/thyroid/retinoid nuclear receptor superfamily (10). However, the mechanisms by which these agents exert their antiproliferative effects are not entirely clear.

We have previously demonstrated that 1,25-VD3 and its noncalciomimetic analog EB1089 induce cell cycle arrest and the accumulation of the cell cycle inhibitor p27 in thyroid cancer cells (11). This effect is not mediated directly through VDR transcriptional regulation of p27, but rather through VDR-mediated regulation of p27 phosphorylation and degradation (11) through the action of the phosphatase and tensin homolog deleted (PTEN) gene. The therapeutic relevance of these findings was underscored recently by the demonstration that systemic 1,25-VD3 administration can significantly arrest thyroid tumor growth and metastasis in vivo (12).

Neoplastic transformation is often characterized by major changes in the organization of the cytoskeleton, decreased cell adhesion, and aberrant adhesion-mediated signaling (13). Disruption of normal cell adhesion may contribute to enhanced migration, proliferation and invasion, favoring metastatic growth. Fibronectin (FN) is an extracellular matrix protein that plays an important role in cell adhesion, migration, tumor invasion and metastasis. Tumor cells are generally characterized by decreased adhesiveness due to failure to deposit stromal FN, followed by rapid proliferation and migration. Reduced FN expression has been noted in transformed cell lines and tumors, including thyroid cancer, where diminished FN expression has been identified particularly at the periphery of invasive tumors (14).

We, therefore, examined in this study the ability of 1,25-VD3 to influence thyroid cancer cell adhesiveness and FN expression. We took advantage of a human thyroid follicular cancer-derived cell line that we have shown to respond to 1,25-VD3 action (11) to determine 1) whether FN is a mediator of 1,25-VD3 action on cell adhesiveness, 2) whether PTEN is responsible for the regulation of FN, and 3) whether FN is critically involved in maintaining organized cellular growth in vivo.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
VD and EB1089 Induce Thyroid Cancer Cell Adhesiveness
We have previously demonstrated that 1,25-VD3 and EB1089 increase p27 levels in WRO follicular-type thyroid carcinoma cells by decreasing phosphorylation and proteasomal degradation of this cyclin-dependent kinase inhibitor (11). This effect was consistent with the presence of functional VDR that we also identified by RT-PCR analysis (data not shown). When treated with 1,25-VD3 or EB1089 under conditions that were effective in arresting WRO cell cycle progression as previously reported (11), cells demonstrated a significant increase in adhesiveness to a collagen I extracellular matrix (Fig. 1Go). We also found that these cells bound to a collagen I matrix twice as efficiently as a collagen IV matrix (not shown) consistent with the higher affinity of FN to collagen I than collagen IV (15).



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Fig. 1. 1,25-VD3 (VD) or Its Analog EB1089 Restore Thyroid Cancer Cell Adhesiveness to an Extracellular Collagen Matrix

WRO follicular-type thyroid cancer cells were treated with VD or EB1089 for 72 h and then were allowed to attach to a collagen I matrix plate, washed, then counted under a stereomicroscope with the aid of imaging software as detailed in Materials and Methods. The bar graph represents the mean + SD of three separate experiments each performed in triplicate wells. P < 0.05 compared with vehicle control is denoted by an asterisk.

 
VD and EB1089 Induce FN Expression
Based on the preference of WRO cells to adhere to a collagen I matrix and previous data implicating VD in the regulation of FN (16), we explored whether the observed effects of 1,25-VD3 on WRO cell adhesiveness were mediated through the extracellular matrix FN protein. Indeed, Western blotting revealed a significant and consistent increase of FN expression when WRO cells were incubated with either 1,25-VD3 or EB1089 (Fig. 2Go).



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Fig. 2. Effect of VD or EB1089 on FN Expression in Thyroid Carcinoma Cells

WRO thyroid cancer cells were treated with VD or EB1089 for 72 h. Cells were lysed, and protein extracts were separated by gel electrophoresis and immunoblotted with antibodies to FN or ß-actin as indicated. A representative gel of each cell type is shown above; the bar graph below depicts the mean + SD of densitometric analysis of Western immunoblotting data from three independent experiments. P < 0.05 compared with vehicle control is denoted by an asterisk.

 
FN Mediates Thyroid Cancer Cell Adhesiveness
To determine whether FN contributes functionally to thyroid cancer cell adhesiveness, we examined the impact of FN down-regulation. Figure 3AGo demonstrates the effect of small interfering RNA (siRNA) on FN expression, and Fig. 3BGo shows the effect of this FN down-regulation on cell adhesiveness. Cells with reduced FN expression demonstrated a reduction of approximately 25% in adhesiveness to a collagen I matrix. Moreover, FN siRNA significantly reduced the effect of 1,25-VD3 treatment on adhesion by approximately 50% (Fig. 3BGo).



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Fig. 3. Effect of siRNA-Mediated FN Down-Regulation on Thyroid Cancer Cell Adhesion

A, WRO cells stably transfected with an expression vector-encoding siRNA sequences to FN (siRNA) and control cells transfected with a vector encoding a scrambled sequence (Control) were subjected to Western immunoblotting for FN detection. Note the selective down-regulation of FN in siRNA-transfected cells. B, WRO cells with FN-down-regulation were subjected to adhesion assays as detailed in Fig. 1Go and Materials and Methods but in the presence or absence of VD. The bar graph represents the mean + SE of three separate experiments performed on two independent clones each in triplicate. P < 0.05 compared with control transfected cells is denoted by an asterisk. Note the diminished adhesiveness displayed by cells with down-regulated FN expression and the reduced ability of VD to maintain cell adhesiveness to the level achieved in control cells.

 
PTEN Induces FN Expression and Cell Adhesiveness
To determine whether the effects of 1,25-VD3 on FN expression in thyroid cancer cells are mediated through the PTEN/phosphoinositol (PI) 3 kinase pathway, we examined the effect of wild-type or phosphatase-mutant forms of PTEN on FN expression and in turn on cell adhesiveness. Transfection of wild-type PTEN significantly enhanced FN expression (Fig. 4AGo). In contrast, transfection of a lipid-phosphatase-inactive (G129E) PTEN mutant or a mutant lacking complete lipid and protein phosphatase activity (C124A) failed to induce FN expression. Instead, both PTEN mutant forms resulted in a reduction in FN expression (Fig. 4AGo). Furthermore, concomitant treatment with 1,25-VD3 of cells transfected with wild-type PTEN did not achieve a greater effect on FN expression compared with either manipulation alone (Fig. 4BGo). Conversely, siRNA-mediated down-regulation of PTEN resulted not only in reduced PTEN expression but also in diminished FN expression (Fig. 4CGo). PTEN down-regulation in these cells either by expression of mutant PTEN forms or by siRNA resulted in diminished cell adhesiveness (Fig. 4DGo). Moreover, cells with down-regulated PTEN expression failed to achieve an adhesiveness level similar to that achieved in 1,25-VD3 treatment of WRO cells transfected with scrambled sequence control siRNA (Fig. 4EGo).



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Fig. 4. Role of PTEN in Regulating FN Expression, Cell Adhesiveness, and VD Responsiveness in WRO Thyroid Carcinoma Cells

A, WRO cells were transfected with expression vectors encoding wild-type or mutant forms of PTEN as indicated. Protein extracts were separated by gel electrophoresis and immunoblotted with an antibody that recognizes FN, PTEN, or ß-actin as indicated. Introduction of wild-type PTEN enhances FN expression. In contrast, the lipid phosphatase-inactive (G129E) PTEN mutant or a mutant PTEN lacking lipid/protein phosphatase activity (C124A) fail to induce FN. P < 0.05 compared with control is denoted by an asterisk. B, VD treatment of cells transfected with PTEN did not achieve a greater effect on FN compared with VD alone. C, WRO cells were transfected with a PTEN siRNA and examined by immunoblotting with an antibody that recognizes PTEN, FN, or ß-actin as indicated. Note the inhibitory effect of PTEN siRNA on PTEN, and the associated reduction in FN expression. D, To examine the effect of PTEN down-regulation on cell adhesiveness, adhesion assays were performed with cells transfected with wild-type PTEN, the lipid phosphatase-inactive G129E PTEN mutant, the PTEN mutant C124A lacking lipid/protein phosphatase activity, or with siRNA to down-regulate PTEN as in panel C. Note the inhibitory effect of mutant forms of PTEN or down-regulation of PTEN on cell adhesiveness. E, WRO cells with down-regulated PTEN were subjected to adhesion assays in the presence or absence of VD treatment. The bar graph represents the mean + SE of two separate transfected clones examined in three separate experiments each performed in triplicate. P < 0.05 compared with control transfected cells is denoted by an asterisk. Note the diminished adhesiveness of cells with down-regulated PTEN and the reduced ability of VD to sustain cell adhesiveness. F, To examine the significance of the PTEN downstream PI3 kinase pathway on cell adhesion, WRO cells were treated with the PI3 kinase inhibitors wortmannin or LY900402 as indicated. Lysates were separated and probed with antisera to FN or ß-actin as indicated. The bar graph represents the mean + SE of three separate experiments each performed in triplicate. Note the enhanced FN expression after PI3 kinase inhibition mimicking the effect of PTEN. The results are representative of three separate experiments, each of which included three wells for each treatment group. P < 0.05 compared with control untreated cells is denoted by an asterisk. G, To determine the effect of PI3 kinase inhibition on WRO cell adhesiveness, adhesion assays were performed after treatment with the PI3 kinase inhibitors wortmannin or LY900402. These inhibitors enhanced cell adhesiveness, consistent with the significance of the PTEN/PI3 kinase pathway in contributing to this cellular function.

 
To further establish the role of the PTEN/PI3 kinase pathway on FN regulation, we examined the impact of the inhibitors wortmannin and LY294002. We have previously demonstrated that both compounds effectively decrease pAkt/protein kinase B and increase p27 accumulation in WRO cells, highlighting the potential importance of the PI3 kinase pathway in mediating VD actions in thyroid cancer cells (11). In the current study, both PI3 kinase inhibitors enhanced basal FN expression (Fig. 4FGo) consistent with the importance of this pathway in regulating FN. Furthermore, the combined treatment of 1,25-VD3 and PI3 kinase inhibitors on FN levels was not appreciably additive (data not shown). That PI3 kinase is critical to thyroid cancer cell adhesiveness was also demonstrated by the impact of wortmannin or LY294002 treatment on adherence to extracellular collagen I matrix (Fig. 4GGo).

FN Restrains Thyroid Cancer Cell Growth and Mediates VD Action in Vivo
To investigate the importance of FN on cell growth in vivo, we examined the pattern of xenografted thyroid cancer cells in severe combined immunodeficient (SCID) mice with or without siRNA-mediated FN down-regulation. Down-regulated loss of FN was associated with accelerated tumor growth (Fig. 5Go). Immunohistochemical examination of these tumors confirmed the persistently down-regulated FN expression, which was accompanied by invasive growth (not shown).



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Fig. 5. Effect of FN Down-Regulation on the Growth and Response to VD Administration in Thyroid Carcinoma Xenografts

WRO cells (5 x106) with down-regulated FN (FN-siRNA) or scrambled sequence (Control) were injected in the flank of 6-wk-old SCID mice as indicated. Treatment consisted of ip injection of 0.75 µg/kg VD or vehicle control (C) three times per week for 3 wk. The indicated tumor volumes (A) and tumor weights (B) represent the mean + SEM of three independent experiments each with five animals in each treatment group. *, P < 0 .05 by paired t test vs. vehicle-treated control. Significant differences were reached by 15 d of treatment and were sustained for the duration of 3-wk experiments.

 
To more specifically examine the impact of FN in mediating 1,25-VD3 antineoplastic action, we compared the effect of 1,25-VD3 treatment on wild-type or siRNA down-regulated FN. As can be seen in Fig. 5Go, cells with down-regulated FN expression failed to demonstrate a comparable growth arrest to that evident in control WRO cells. During monitoring, tumor volume was consistently higher in small interfering FN (siFN)-xenografted cells than in controls and the effects of 1,25-VD3 resulted in reduced volumes in both groups that were detectable by 15 d of treatment. At the end of the 3 wk of treatment, tumors were resected and tumor weight was reduced by 1,25-VD3 by approximately 50% in control cells but only by approximately 22% in siFN xenografted cells; tumor volume was reduced by 36% in controls vs. 24% in siFN cells (P = 0.018). This 1,25-VD3 treatment was not associated with any significant toxicity as evidenced from serum calcium levels or animal weights as previously described (12). Taken together, these findings provide strong evidence for a constitutive role for FN in maintaining thyroid cancer cell adhesiveness and in, at least partially, mediating the suppressive functions of 1,25-VD3 on these cells.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
It is becoming increasingly evident that diminished cell-cell and cell-matrix adhesiveness represents a morphologic hallmark of neoplasia (17). This disruption can be achieved by down-regulation of the cadherin/catenin network linked to the cytoskeletal framework or alternatively by activation of signaling pathways that prevent the assembly of adherens junctions. Evidence in favor of the former in thyroid cancer is presented by studies of thyroid cancer cell lines and primary tumors that have confirmed dysregulated ß-catenin signaling (18, 19).

We and others (11, 20) have previously shown that 1,25-VD3 and its noncalciomimetic analog EB1089 can inhibit thyroid carcinoma cell growth by inducing cell cycle arrest in human thyroid papillary and follicular carcinoma cell lines. In this study, we explored alternative mechanisms for the growth inhibitory effects of VD compounds. We focused on FN as a potential target of VD-mediated cell adhesiveness for a number of reasons. Firstly, FN is an established extracellular matrix component contributing to cell adhesion that is frequently diminished or reduced in solid neoplasms (21). In particular, diminished stromal FN staining has been associated with increased metastatic potential in invasive breast carcinoma (22) as well as thyroid cancer (14, 23, 24). The diminished stromal FN staining has been found to be most evident at the periphery of invasive tumors (14). Secondly, a VD response element in the FN gene has been identified to bind VDR homodimers (25). Consistent with this prediction, VD has been shown to stimulate FN in human osteosarcoma MG-63 cells (16). Overexpression of FN suppresses human fibrosarcoma HT1080 cell growth (26). Interestingly, reattachment of suspended keratinocytes to FN leads to restoration of VDR expression, whereas VDR expression is reduced by disruption of the actin cytoskeleton (27). These findings predicted a possible functional network between VD responsiveness and cell adhesion through FN.

In the current study, we show for the first time that 1,25-VD3 and related compounds can enhance FN expression and restore adhesiveness of thyroid follicular carcinoma cells. Further evidence for the presence of functional VDR in thyroid cancers is demonstrated by their responses to both VD compounds in terms of cell cycle arrest and p27 accumulation (11). In particular, we noted that 1,25-VD3 and its analogs were potent activators of the PTEN/PI3 kinase pathway in thyroid cancer cells (11). Given the documented VD response element in the FN promoter (25), the current data on the contribution of PTEN/PI3 kinase in inducing FN highlight the contribution of at least one additional signaling pathway in regulating this gene.

Mutations and/or deletions of PTEN have been implicated in a number of malignancies, including thyroid carcinoma (28, 29). Mice heterozygous for a PTEN deletion (+/–) develop multiorgan neoplasia including thyroid cancer (30, 31, 32), highlighting the importance of intact PTEN function in normal thyroid cell homeostasis. Indeed, down-regulation of PTEN has been noted in a subset of human sporadic thyroid tumors (33, 34, 35) and thyroid carcinoma cell lines (36, 37). Consistent with these findings, we had previously shown that VD compounds can restore PTEN expression in WRO cells but do not significantly enhance endogenous PTEN levels or activity in papillary thyroid cancer NPA cells (11). We now demonstrate that 1,25-VD3 can also restore FN expression in WRO cells. Multiple lines of evidence support the notion that the PTEN/PI3 kinase pathway is critical for mediating 1,25-VD3 actions in these cells (11, 38). Firstly, introduction of wild-type, but not phosphatase-mutant forms of PTEN, was effective in positively influencing FN expression. Conversely, siRNA-mediated down-regulation of PTEN diminished FN expression as well as cell adhesiveness. Further support for the importance of the PTEN/PI3 kinase pathway in mediating VD regulation of FN was derived from pharmacologic PI3 kinase inhibition, which also enhanced FN expression. This FN up-regulation was associated with enhanced cell adhesiveness. Finally, down-regulated PTEN resulted in reduction of the adhesive effect of 1,25-VD3 on thyroid cancer cell growth. Taken together, these findings provide evidence supporting the critical contribution of PTEN in mediating growth suppressive functions in thyroid cancer. We also demonstrate that FN is an important target of PTEN-mediated cell adhesiveness. That PTEN is a mediator of 1,25-VD3 action is functionally consistent with the well-recognized effects of PTEN not only on cell proliferation but also on control of cell invasion and focal adhesions (17).

Primary human thyroid cancers have been shown to exhibit up-regulation of FN, a phenomenon that was identified by cDNA microarray studies (39, 40) and corroborated by immunohistochemical studies (41). It remains unclear how much of this up-regulation is due to stromal cell production (42). Up-regulation of FN may represent a compensatory mechanism induced to enhance tumor cell adhesiveness in differentiated neoplastic cells. Consistent with this concept, tumor cells in metastatic thyroid cancers are more likely to be negative for FN at the periphery of invading tumor than those in nonmetastatic cases (14), suggesting that FN may represent a target for the treatment of the more invasive and metastatic thyroid cancers.

The full spectrum of mechanisms by which VD compounds can exert antineoplastic actions remains to be established. Our data indicate that 1,25-VD3 and noncalcimimetic analogs can impose proadhesive effects on thyroid carcinoma cells. The implications of these properties are further underscored by the effects of 1,25-VD3 that have recently been suggested to significantly reduce thyroid carcinoma growth and invasiveness in an orthotopic mouse model (12). In this model, 1,25-VD3 treatment reduces the frequency and extent of invasive and metastatic thyroid cancerous growth. Nevertheless, noncalciomimetic VD analogs such as EB1089 may represent more suitable therapeutic alternatives in the treatment of thyroid cancer. The current studies provide new evidence that the actions of VD compounds on cell adhesion can be at least partially ascribed to their effects on FN. Indeed, down-regulation of FN was associated not only with more invasive growth in vivo but also with a relative insensitivity to the antineoplastic actions of VD. These findings establish FN and PTEN as important factors in maintaining thyroid cell adhesiveness and in determining some VD actions. They also suggest that the up-regulation of FN noted in gene profiling studies may represent a favorable tissue response to neoplastic transformation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Compounds
1,25-VD3 and its noncalciomimetic analog EB1089 (22, 24-diene-24a, 26a, 27a-trihomo-1{alpha}, 25-dihydroxy-vitamin D3) were kindly provided by LEO Pharmaceutical Products (Ballerup, Denmark) and were used as previously described (11, 38). Inhibition of PI-3 kinase was achieved using the selective agents wortmannin 0.2 µM (Sigma, St. Louis, MO) or LY900402 20 µM (New England Biolabs, Beverly, MA) as previously described (11, 38).

Cell Culture
The human thyroid follicular WRO carcinoma cell line, originally established by Dr. G. Juillard (UCLA, Los Angeles, CA), was kindly provided by Dr. J. Fagin (Cincinnati, OH). Cells were maintained in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1x nonessential amino acids and antibiotics.

RT-PCR
Total RNA was reverse transcribed and PCR amplified using forward primers for human VDR as follows: 5'-GAC TTT GAC CGG AAC GTG CC-3' (exon 2) and the reverse primer: 5'-CAT CAT GCC GAT GTC CAC AC-3' (exon 3) as described previously (43).

PTEN Plasmids
GFP-tagged vectors encoding wild-type PTEN, the lipid phosphatase inactive mutant G129E, and the dual lipid/protein phosphatase-inactive mutant C124A were generously provided by Dr. K. Yamada (National Institutes of Health, Bethesda, MD).

FN and PTEN siRNA Plasmid Construction
The target sequence for FN down-regulation AAC AAA TCT CCT GCC TGG TAC was aligned to the human genome database in a basic local alignment and search tool (BLAST) to exclude homology with unrelated genes. The siRNA oligonucleotide templates were as follows: top strand, GAT CCG CAA ATC TCC TGC CTG GTA CTC TGC AGG AGT ACC AGG CAG GAG ATT TGT TTT TTG GAA A; bottom strand, AGC TTT TCC AAA AAA CAA ATC TCC TGC CTG GTA CTC CTG CAG AGT ACC AGG CAG GAG ATT TGC G). These were prepared according to the manufacturer’s instructions (Ambion Inc., Austin, TX). After annealing, the duplexes were ligated into pSilencer 2.1-U6 neo vector at the BamHI/HindIII sites. The products were transformed into DH5{alpha} competent cells. Ampicillin-resistant colonies were selected, identified by restriction digestion using PstI, and further confirmed by DNA sequencing.

PTEN siRNA and the respective mismatch control in the U6+2 vector were gifts from Dr. J. Kaufmann (44)

Transfections
Transfections were performed using LipofectAMINE plus (Invitrogen Life Technologies, Carlsbad, CA). Cells were cultured in 6-cm plates until 60–70% confluent and transfected with 5 µg of the PTEN plasmid. Transfection efficiency was evaluated by GFP fluorescence. For siRNA studies, 1 d after transfection, cells were placed into selection medium containing 1 mg/ml G418 (Invitrogen Life Technologies) and individual geneticin-resistant colonies were screened by Western blotting. Control cells were transfected with scrambled sequences (ssRNA). Successful expression was determined by Western blotting and protein down-regulation was confirmed by demonstrating at least a 70% reduction in signal intensity compared with control ssRNA-transfected cells. At least two independent stably transfected clones for each sequence were used for subsequent studies.

Western Blotting
After transfections and/or incubations with 1,25-VD3, EB1089, or vehicle control, cells were lysed in RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 100 µg/ml phenylmethylsulfonyl fluoride, aprotinin, and sodium orthovanadate in PBS). Total cell lysates were incubated on ice for 30 min followed by microcentrifugation at 10,000 x g for 10 min at 4 C. Fifty micrograms of protein were separated by 10 or 12% SDS-PAGE and transferred onto nitrocellulose membranes, which were blocked in 5% nonfat milk and 0.1% Tween 20 in TBS 20 mM Tris-Cl and 500 mM NaCl (pH 7.5) for 1 h and incubated overnight at 4 C with the following primary antibodies: FN (1:5000, Transduction Laboratories, Lexington, KY), PTEN (1:100, Chemicon International, Inc., Temecula, CA), and ß-actin (1:500, Sigma, Oakville, Ontario, Canada). After washing, membranes were incubated for 1 h at room temperature with peroxidase conjugated antimouse IgG secondary antibody (1:2000, Santa Cruz Biotechnology Inc., Santa Cruz, CA). Protein bands were visualized by chemiluminescence as described by the supplier (Amersham, Oakville, Ontario, Canada) and band intensities were quantified by densitometric scanning.

Adhesion Assay
Cells were incubated with 1,25-VD3 or EB1089 (10–7 M) for 24–72 h. After trypsinization, 1 x 105 cells were plated in each well of 24-well plates coated with collagen I or collagen IV as indicated (Becton Dickinson, Bedford, MA), incubated for 1 h, then rinsed with PBS four times. The remaining cells in each well were fixed in 10% buffered formalin for 20 min at room temperature, washed in PBS, and the entire field counted with a stereomicroscope.

Another approach used GFP transfection; adherent GFP-positive cells were visualized using a fluorescence stereomicroscope (Leica, Heidelberg, Germany) before and after washing to determine the percent of cells that were adherent.

All images were recorded and analyzed using Image-Pro Plus Software (version 4.5; Media Cybernetics Inc., Silver Spring, MD). Screen captures were taken to match the area of interest to the exported data for analysis with Excel.

Mouse Model of Thyroid Cancer
Human thyroid follicular carcinoma-derived WRO cells were implanted in the flank of 4- to 5-wk-old SCID mice at a concentration of 5 x 106 cells and monitored for the development of tumor growth. Toxicity was monitored by assessing body weight, serum calcium, and overall health status as previously described (12).

1,25-VD3 Therapy
Animals were treated with 1,25-VD3 0.75 µg/kg or vehicle ip three times per week for 21 d as previously described (12). Treatments commenced 5 d after tumor cells were implanted. Animal handling and treatment protocols were approved by the Ontario Cancer Institute Animal Care and Utilization Committee.

Analysis of Tumor Growth
Tumor growth and volume were measured using calipers every 5 d (tumor volume in mm3 = tumor width x tumor width x tumor length /2). Mice were killed 21 d after tumor cell implantation and the tumors were excised and measured. Excised tumor tissue was fixed in formalin, embedded in paraffin, and examined by light microscopy and immunohistochemistry for FN and thyroglobulin (12). Complete necropsies were performed with survey of all tissues for metastatic disease grossly and by light microscopy as previously described (12).

Statistical Analysis
Data are presented as mean ± SE. Differences were assessed by Student’s paired t test. Significance level was assigned at P < 0.05.


    FOOTNOTES
 
This work was supported by the Toronto Medical Laboratories and the Rita Banach Thyroid Cancer Research Fund.

First Published Online May 12, 2005

Abbreviations: FN, Fibronectin; PI3 kinase, phosphoinositol 3 kinase; PTEN, phosphatase and tensin homolog deleted; SCID, severe combined immunodeficient; siFN, small interfering FN; siRNA, small interfering RNA; VD, vitamin D; 1,25-VD3, 1{alpha},25-dihydroxyvitamin D3; VDR, VD receptor.

Received for publication March 8, 2005. Accepted for publication May 2, 2005.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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