An Adenovirus Carrying the Rat Protein Tyrosine Phosphatase η Suppresses the Growth of Human Thyroid Carcinoma Cell Lines in Vitro and in Vivo¹ Free

Thyroid tumors include a wide spectrum of lesions that differ in phenotypic characteristics and biological behavior: benign adenomas, differentiated carcinomas and undifferentiated ATCs³ (1). Treatment with radioactive iodide is very effective in cases of differentiated carcinomas. Conversely, undifferentiated carcinomas are refractory to chemo- and radiotherapy, and are among the most lethal human neoplasms (2). Therefore, ATCs are an appropriate target for novel therapeutic approaches.

The vector-mediated transduction of genes able to arrest tumor growth rate, defined as “cancer gene therapy,” may be a useful alternative or complementary strategy to conventional therapies. Gene therapy has been successful in experimental models of thyroid carcinoma. In fact, suppression of the HMGA1 proteins leads to the apoptotic death of two human thyroid carcinoma cell lines (3). Similarly, transduction of adenovirus-mediated p53 inhibited thyroid carcinoma cell growth (4). We recently showed that an E1B M_r 55,000 gene-defective adenovirus (ONYX-015), which replicates only in cells with impaired p53 function, killed thyroid carcinoma cells. Furthermore, the ONYX-015 virus and two antineoplastic drugs (doxorubicin and paclitaxel) acted synergistically (5).

These results prompted us to investigate the potential of the r-PTPη gene for cancer therapy. This gene, isolated by our group, encodes a receptor-type tyrosine phosphatase protein of which the expression is reduced drastically in rat thyroid cells transformed by acute murine retroviruses (6). In addition, the expression of HPTPη/DEP-1, the human homologue of r-PTPη, is down-regulated in human thyroid carcinomas (7). The re-expression of the r-PTPη gene in highly malignant rat thyroid cells transformed by retroviruses carrying the v-mos and v-ras-Ki oncogenes suppresses their malignant phenotype. The level of the cyclin-dependent kinase inhibitor p27^Kip1 protein was increased in the r-PTPη-transfected cells because of the decreased proteasome-dependent degradation rate. Because suppression of p27^Kip1 protein synthesis blocked the inhibition of growth induced by r-PTPη, we proposed that r-PTPη tumor-suppressor activity is mediated by p27^Kip1 protein stabilization, which, in turn, depends on the r-PTPη-mediated inhibition of ERK1/2 activation induced by the v-ras-Ki and v-mos oncogenes (7). The mouse homologue of r-PTPη/DEP-1 (ptprj) has been implicated recently in susceptibility to mouse colon cancer (8). Allelic imbalance in loss of heterozygosity and missense mutations of this gene are frequent in human colon, lung, and breast cancers (8).

The aim of our study was to generate an adenovirus carrying the full-length r-PTPη cDNA (Ad-r-PTPη) to examine whether r-PTPη/DEP-1 overexpression could be effective in treating human thyroid carcinomas. We found that the Ad-r-PTPη virus inhibited thyroid carcinoma cell growth, and that growth inhibition was associated with an increased p27^Kip1 protein level and reduced ERK1/2 activity. Moreover, r-PTPη overexpression reduced the phosphorylation level of PLCγ1, a substrate of DEP-1 (9). Finally, the growth of xenograft tumors induced in athymic mice by the injection of ARO cells was drastically reduced by Ad-r-PTPη treatment.

The BC-PAP, NIM, NPA, and TPC-1 cells were derived from human thyroid papillary carcinomas. WRO were derived from human thyroid follicular carcinoma. ARO and FB-1 were derived from human anaplastic carcinomas (10, 11). All of the cell lines were grown in DMEM supplemented with 10% fetal bovine serum and ampicillin/streptomycin antibiotics at 37°C and 5% CO₂.

Cells were washed once in cold PBS and lysed in a buffer containing 50 mm Tris-HCl (pH 7.5), 1% (v/v) NP40, 150 mm NaCl, complete protease inhibitors (Roche), 50 mm NaF, and 1 mm sodium vanadate. Lysates were clarified by centrifugation at 10,000 × g for 15 min, and the supernatant was collected. Protein concentration was determined by a modified Bradford assay (Bio-Rad). Five-hundred μg of proteins were immunoprecipitated by using 1 μg of anti-PLCγ1 antibodies and 20 μl of protein G-agarose (Upstate Biotechnology). Immunoprecipitates were washed three times in lysis buffer. Total proteins or immunoprecipitates were separated on 4–20% polyacrylamide gels and transferred to polyvinylidene difluoride filter membranes (Immobilon-P; Millipore). Membranes were blocked in 5% nonfat dry milk (1% BSA for phosphotyrosine detection), incubated with primary antibodies, detected by the appropriate secondary antibodies, and revealed with an enhanced chemiluminescence system (Santa Cruz Biotechnology). The primary antibodies used were: polyclonal rabbit antibodies generated against the intracellular region of r-PTPη fused to GST (7); anti-pERK, anti-ERK, anti-p27, anti-PLCγ1, and anti-γ-tubulin from Santa Cruz Biotechnology; and antiphosphotyrosine from Transduction Laboratories.

The r-PTPη insert was prepared by digesting the cDNA with PauI and EcoRI at the 5′ and 3′ end, respectively. Blunt ends were generated with Klenow DNA polymerase, and the cDNA fragment was cloned into the transfer vector pQBI/AdCMV5-GFP (Qbiogene) linearized previously with BglII and then treated with Klenow to generate compatible blunt ends.

The pQBI/AdCMV5-GFP/r-PTPη construct was cotransfected with the E1/E3 deleted Ad5 backbone viral DNA. Viral plaques were screened for the presence of the r-PTPη protein by Western blot with specific r-PTPη antibodies. One positive clone was plaque-purified and amplified on 293 cells. After freeze/thaw cycles, the adenoviruses in the supernatant were purified on two successive cesium chloride gradients. The recombinant adenovirus was titered by the TCID50 method and aliquoted. Virus stocks were stored at −80°C. An adenovirus carrying GFP under the control of a CMV promoter (Ad5.CMV-GFP; Qbiogene) was used as a control.

Cells (5 × 10⁴) were seeded in a six-well plate. After 24 h, cells were infected at a MOI of 100 with Ad-r-PTPη or Ad-GFP for 90 min using 500 μl of infection medium (DMEM + 2% fetal bovine serum) at 37°C in a 5% CO₂ incubator. The optimal MOI for each cell line was determined in pilot experiments with Ad-GFP. At an MOI of 100, the cell lines used became GFP-positive without manifesting signs of toxicity. Infected cells were harvested and counted daily using a hematocytometric chamber. Three independent experiments were performed for each cell line.

The percentage of cell growth inhibition was calculated with the formula: (x − y)/x (x is the difference between the mean cell count 4 days after Ad-GFP infection and the number of cells plated; y is the difference between the mean cell count 4 days after Ad-r-PTPη infection and the number of cells plated).

ARO cells (1 × 10⁶) uninfected or infected at an MOI of 100 with Ad-r-PTPη or Ad-GFP were injected s.c. into athymic mice. Tumor volumes and weights were evaluated 30 days later, after which the mice were killed. Tumor volumes (V) were calculated by the rotational ellipsoid formula: V = A × B²/2 (A = axial diameter; B = rotational diameter). None of the mice showed signs of wasting or other signs of toxicity.

Expression of DEP-1 was examined in seven cell lines derived from ATCs (ARO and FB-1), from a thyroid carcinoma with a follicular histotype (WRO), and from thyroid carcinomas with a papillary histotype (BC-PAP, NIM, NPA, and TPC-1). Because DEP-1 expression is regulated by cell density (12), DEP-1 protein levels being higher in dense than in sparse cultures, we evaluated DEP-1 protein levels by Western blot using protein extracts from cells at 50% confluency and at full confluency. As shown in Fig. 1, two protein bands were detected, which probably represent differently glycosylated DEP-1 forms, as observed by others (12). DEP-1 protein levels were lower in all of the thyroid carcinoma cell lines than in thyroid gland. In particular, DEP-1 expression was very low in BC-PAP and ARO cells. Moreover, in some cell lines (ARO, NIM, and WRO), DEP-1 expression was not influenced by cell density, whereas in other cell lines (BC-PAP, FB-1, NPA, and TPC-1) DEP-1 protein levels significantly increased when the cells reached full confluency. The differences in DEP-1 expression consequent to changes in cell density in the thyroid carcinoma cells did not depend on the absolute protein levels in the various cell lines.

To evaluate the therapeutic potential of r-PTPη as a therapeutic gene, we generated a replication defective adenovirus carrying r-PTPη cDNA (Ad-r-PTPη). The cDNA was cloned into the transfer vector pQBI AdCMV5-GFP under the transcriptional control of a CMV promoter. This vector also carries the GFP reporter gene of which the expression is controlled by another independent CMV promoter (Fig. 2,A). The recombinant adenovirus was then obtained as described under “Materials and Methods.” The thyroid cell lines ARO, FB-1, NIM, and TPC-1 were infected with the Ad-r-PTPη virus at an MOI of 100. Significant r-PTPη protein levels were detected by Western blot analysis in all of the cell lysates collected at different time points after Ad-r-PTPη infection. r-PTPη was overexpressed both in FB-1 cells that express significant protein levels of endogenous DEP-1 and in ARO cells that express very low DEP-1 protein levels (Fig. 2 B). Protein r-PTPη was not overexpressed in the same carcinoma cells infected with the control virus (Ad-GFP). As expected, the protein band of r-PTPη has a molecular weight lower than that of endogenous DEP-1 (6).

Subsequently, we evaluated the growth rate of four thyroid carcinoma cells infected with Ad-r-PTPη and control adenovirus (Ad-GFP). As shown in Fig. 3, the cell growth rate was reduced significantly in ARO, FB-1, NIM, and TPC-1 cell lines infected with Ad-r-PTPη compared with the same cells infected with the control virus. The percentage of growth inhibition, calculated as described under “Materials and Methods,” 4 days after infection was 85% in ARO cells, 73% in FB-1 cells, 62% in NIM cells, and 77% in TPC-1 cells.

Because we demonstrated that r-PTPη inhibits the growth of malignant rat thyroid cell lines by down-regulating ERK1/2 activity and by up-regulating p27^kip1 (7), we evaluated phosphorylated ERK1/2 and p27^kip1 protein levels in human thyroid cell lines infected with Ad-r-PTPη. As shown in Fig. 4 A, Western blot analysis demonstrated a decrease of phosphorylated ERK1/2 and an increase of p27^kip1 protein levels in Ad-r-PTPη-infected cells compared with cells infected with the control adenovirus.

To verify whether r-PTPη acts on the same substrates as its human homologue DEP-1 in human thyroid cell lines, we evaluated r-PTPη activity on PLCγ1, a DEP-1 substrate (9). We immunoprecipitated protein extracts of the infected ARO cells with specific antibodies that recognize human PLCγ1, and then we detected the immunoprecipitates with antiphosphotyrosine antibodies. As shown in Fig. 4 B, tyrosine phosphorylated PLCγ1 was detectable in Ad-GFP-infected ARO cells but not in the ARO cells infected with the Ad-r-PTPη virus. This result demonstrates that r-PTPη is able to dephosphorylate DEP-1 substrates.

Finally, to evaluate the efficacy of Ad-r-PTPη in inhibiting tumor growth in vivo, we examined the effect of Ad-r-PTPη in ARO cells that generate tumors when injected s.c. into athymic mice. ARO cells uninfected or infected with either Ad-r-PTPη or Ad-GFP at an MOI of 100 were inoculated into 24 mice (8 mice per group). Thirty days later, the average tumor size in mice injected with Ad-r-PTPη-infected cells was ∼8-fold smaller than those in the mice injected with uninfected cells or Ad-GFP-infected cells (Fig. 5).

This study shows that restoration and/or overexpression of the r-PTPη gene in thyroid carcinoma may represent a novel treatment approach to human thyroid carcinomas. The undifferentiated anaplastic carcinoma could be a particularly appropriate target for innovative gene therapy, being a very aggressive human neoplasia that is resistant to any known therapeutic approach (2).

We found that infection of thyroid cells with a recombinant adenovirus carrying the full-length cDNA of r-PTPη (Ad-r-PTPη) resulted in the synthesis of a relevant amount of r-PTPη protein, and that r-PTPη gene overexpression inhibited the growth of all of the thyroid carcinoma cell lines tested. The inhibitory effect of Ad-r-PTPη was strongest in ARO cells, which express very low levels of endogenous DEP-1 protein, and lowest in NIM cells, in which endogenous DEP-1 protein levels are unaffected by cell confluency. Therefore, it can be speculated that cells in which DEP-1 expression increases with cell density could be more sensitive to the inhibitory effect of r-PTPη. Furthermore, the transduction of r-PTPη in malignant human thyroid cells resulted in inhibition of tumor growth in athymic mice.

We show that cell growth inhibition correlates with decreased ERK1/2 activity and increased p27^kip1 protein levels. These results are consistent with our previous data on rat thyroid malignant cells (7) and with the critical role played by p27^kip1 in inhibiting the growth of human thyroid cell lines (13). Furthermore, r-PTPη overexpression induces the tyrosine dephosphorylation of PLCγ1, a DEP-1 substrate (8). PLCγ1, when activated by tyrosine phosphorylation, increases the growth and invasiveness of various cancer cell types (14, 15). As a consequence, PLCγ1 dephosphorylation by r-PTPη may inhibit PLCγ1 activity. This in turn suggests that Ad-r-PTPη might be viable for the treatment of tumors with elevated PLCγ1 activity. Data on cell growth inhibition consequent to DEP-1 and r-PTPη overexpression in breast (16) and pancreatic⁴ carcinoma cell lines, suggest that Ad-r-PTPη may be useful for cancers other than thyroid carcinomas. Support for this concept comes from recent findings showing that DEP-1 is deleted frequently in human colon, lung, and breast cancers (8).

Given the finding that r-PTPη re-expression is associated with partial restoration of differentiated thyroid functions (7), one may envisage r-PTPη-based gene therapy combined with radioactive iodine treatment in cases of ATC. In fact, being undifferentiated, these tumors cannot trap iodide and are therefore resistant to iodine treatment. In these optics, the differentiation state of Ad-r-PTPη-infected human cell lines deserves further study. The possibility of synergism between the Ad-r-PTPη virus and conventional antineoplastic drugs should also be investigated.

In conclusion, restoration of normal r-PTPη activity in human thyroid carcinoma cells may represent a novel therapeutic approach to thyroid cancer therapy.

We thank the Associazione Partenopea per la Ricerche Oncologiche (APRO) for its support. We also thank Jean Ann Gilder for editing the text.