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Notch1 is a multifunctional transmembrane receptor that regulates cellular differentiation, development, proliferation, and survival in a variety of contexts. We have previously shown that Notch1 may function as a tumor suppressor and that histone deacetylase (HDAC) inhibitors can induce Notch1 expression in some endocrine cancers. Here, we showed that while there was minimal Notch1 expression in follicular thyroid cancer FTC236 and papillary thyroid cancer DRO cells, transfection of constitutive Notch1 plasmid into these cells led to growth inhibition, down-regulation of cyclin D1 and up-regulation of p21. Treatment of FTC236 cells with HDAC inhibitors valproic acid (VPA, 1–4 mM) or Suberoyl bishydroxamic acid (SBHA, 10–30 µM) induced functional Notch1 protein expression and suppressed cells growth in a dose-dependent manner. Notch1 siRNA interference blocked the antiproliferative effect of HDAC inhibitors. Western blot analysis revealed reduction of cyclin D1 and increase of p21 in HDAC inhibitors treated cells. These results indicate that HDAC inhibitors activate Notch1 signaling in thyroid cancer cells and lead to suppression of proliferation by cell cycle arrest. Our findings provide the first documentation of the role of Notch1 signaling as a tumor suppressor in DRO and FTC236 cells, suggest that Notch1 activation may be a potential therapeutic target for papillary and follicular thyroid cancers.
Papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC) are the two most common thyroid cancers. They account for 90–95% of all thyroid malignancies and are most often seen in patients over forty years of age (1). The tumor usually presents as an asymptomatic solitary intrathyroid nodule. In patients with FTC, distant metastases at the time of diagnosis are reported in 11–20% of patients (2). FTC tends to metastasize hematogeneously to lung and bone while PTC is commonly metastatic to regional lymph nodes and lung. Synchronous lung metastasis in both types of cancers has been reported in about 20% of cases, with a mean age at presentation of over fifty years (3).
Despite classified collectively as well differentiated thyroid carcinoma, PTC and FTC have distinct clinicopathologic features, biologic behavior, and clinical outcome (4,5). FTC is generally considered to be a more aggressive tumor than PTC and is associated with a worse prognosis. Patients with FTC often present with more advanced stage diseases and a higher incidence of distant metastases because of the propensity of vascular invasion. In contrast, lymph node metastasis is quite uncommon in FTC with an average incidence of <10%. Thyroid cancer management has not changed substantially during the past decades. Treatment is based on total thyroidectomy, ablative doses of radioiodine and suppressive treatment. The follow-up is based on measuring serum thyroglobulin (Tg) and imaging with radioiodine scans.
Although the prognosis of patients with differentiated thyroid cancer is good, and most patients survive long disease-free intervals after appropriate thyroid surgery (6,7)and, when necessary, radioiodine I131 therapy (8), in a subset of patients, thyroid cells lack or lose the capacity to concentrate radioiodine over time. Lack of radioiodine uptake in thyroid cancer is usually associated with increased growth rate and larger tumor load. This is seen in approximately 50% of patients with distant metastases. Therefore, alternative strategies for the treatment of the metastatic papillary and follicular thyroid cancers are needed.
Notch1 is a multifunctional transmembrane receptor that regulates cellular differentiation, development, proliferation, and survival in a variety of contexts (9–11). In human cancer cells, Notch1 acts as either a tumor suppressor or an oncogene with its role being dependent upon its cellular context (12). The oncogenic role of Notch1 was identified in many types of cancer, including pancreatic cancer, colon cancer, non-small cell lung cancer, cervical cancer, renal cell carcinoma, and several lymphomas (13–15). However, we have shown the absence of active Notch1 in neuroendocrine tumors (NETs), and that over expression of Notch1 leads to tumor growth suppression (16). Moreover, the degree of growth inhibition is directly proportional to the amount of Notch1 present. Histone deacetylase (HDAC) inhibitors are a class of potent antineoplastic agents that induce differentiation, growth arrest, and apoptosis of transformed cells. Very recently, we have shown that treatment of NET cells with the HDAC inhibitors valproic acid (VPA) and suberoyl bishydroxamic acid (SBHA) led to growth suppression associated with Notch1 activation (17–20). Moreover, a significant reduction in NET markers is mediated by Notch1 activation in HDAC inhibitors treated cells. These findings indicate that activation of Notch1 signaling may have a therapeutic role on treating NETs.
In this study we report that Notch1 is highly expressed in normal human thyroid tissue but markedly downregulated in metastatic thyroid cancer specimens. Simiarly, Notch1 signaling is minimal in metastatic PTC and FTC cells. Treatment of FTC cells with HDAC inhibitors VPA and SBHA resulted in a dose dependent increase in Notch1 protein. Furthermore, a dose dependent reduction in growth was also observed. Transfection of siRNA against Notch1 blocked the antiproliferative effect of HDAC inhibitors. These findings provide the first documentation of the role of Notch1 signaling as a tumor suppressor in thyroid cancer cells; suggest that Notch1 activation may be a potential therapeutic target for papillary and follicular thyroid cancers.
DRO and FTC236 cells provided by Dr. Fiemu Nwariaku (UT Southwestern, Dallas, TX) were maintained in Dulbecco’s Modified Eagle/Ham’s F-12 (1:1; Invitrogen, Carlsbad, CA) medium supplemented with 10% fetal calf serum, 0.01 U/mL thyroid-stimulating hormone, 10 µg/mL insulin (Sigma-Aldrich, St. Louis, MO), 50 µg/mL penicillin/streptomycin ( Invitrogen). WI-38 cells, a human embryonic lung fibroblast, were maintained in minimal essential medium (Eagle) with 10% fetal calf serum.
A constitutive NICD plasmid (TAN1, kindly provided by Dr. Douglas W. Ball, Johns Hopkins University) or a vector plasmid LNCX1 as control was transiently transfected into DRO and FTC 236 cells using Lipofectamine 2000 (Invitrogen, San Diego, CA) as described previously (16). Next day, the cells were trypsinized, counted, and plated in equal amounts (5,000 cells per well) onto 24-well plates for a cell proliferation assay.
Cell proliferation was measured by the methylthiazolyldiphenyl-tetrazolium bromide (MTT; Sigma-Aldrich) rapid colorimetric assay as previously described (16). Briefly, 5000 cells were seeded in quadruplicate on 24-well plates and incubated overnight under standard conditions to allow cells attachment. The cells were then treated with VPA (Sigma-Aldrich) in concentrations of 0–4 mM or SBHA (Sigma-Aldrich) in concentrations of 0–30 µM and incubated for up to 6 days with media changes every 2 days. The MTT assay was performed by replacing the standard medium with 250 µl of serum-free medium containing MTT (0.5 mg/ml) and incubating at 37°C for 3 hours. After incubation, 750 µl of dimethyl sulfoxide (DMSO, Fisher Scientific, Pittsburgh, PA) was added to each well and mixed thoroughly. The plates were then measured at 540 nm using a spectrophotometer (µQuant; Bio-Tek Instruments, Winooski, VT).
Whole cell lysates were prepared as previously described (16). Total protein concentrations were determined using a bicinchoninic acid assay kit (BCA; Pierce Biotechnology, Rockford, IL). Denatured cellular extracts (30–50µg) were resolved by SDS-PAGE, transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA), blocked in milk and incubated with appropriate antibodies. The primary antibodies dilutions were: 1:2,000 for Notch1 (Santa Cruz Biotechnology, Santa Cruz, CA), cyclin D1, p21, p15, p27 and Acetyl-Histone H4 (AH4) (Cell Signaling Technology, Danvers, MA); and 1:10,000 for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Trevigen, Gaithersburg, MD). Horseradish peroxidase conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (Cell Signaling Technology) were used depending on the source of the primary antibody. For visualization of the protein signal, Immunstar (Bio-Rad Laboratories, Hercules, CA) or SuperSignal West Femto (Pierce Biotechnology) kits were used per the manufacturer's instructions.
Cells were transiently transfected with luciferase constructs as previously described (16). Wild-type (4xwtCBF1Luc; 2µg) CBF-1 luciferase reporter plasmids (a generous gift from Dr. Diane Hayward, Johns Hopkins University) were cotransfected with cytomegalovirus ß-galactosidase (CMV-ß-gal; 0.5 µg) (21). After transfection, cells were treated with VPA (2 mM and 4 mM), SBHA (20 µM) or DMSO as control for 48 hours. Cells were harvested and lysed, luciferase and ß-galactosidase assays (Promega, Madison, WI) were performed in accordance with the manufacturer's instructions. Luciferase levels were measured using a Monolight 2010 Luminometer (Analytical Luminescence Laboratory, San Diego, CA). Luciferase activity was expressed relative to ß- galactosidase activity.
To determine the effect of Notch1 on cellular proliferation, siRNA against Notch1 and nonspecific siRNA (Santa Cruz Biotechnology) were transfected into thyroid cancer cells using Lipofectamine 2000 (Invitrogen) as described previously (18). The next day, the cells were trypsinized, counted, and plated in equal amounts (5,000 cells per well) onto 24-well plates. On the following day, the cells were treated with DMSO as control or VPA (4 mM) or SBHA (30 µM), MTT assay was performed on day 2, 4 and 6.
Human normal thyroid tissues and thyroid cancer specimens were obtained through a human subjects institutional review board approved protocol. Samples were processed for total protein as previously described (16). Total proteins were analyzed for Notch1 and GAPDH expression by Western analysis as described above.
Analysis of variance with Bonferroni post hoc testing was performed using a statistical analysis software package (SPSS version 10.0, SPSS, Chicago, IL). A p-value of <0.05 was considered significant.
Normal human thyroid tissue and thyroid cancer samples were analyzed for the active Notch1 (NICD) protein by Western blot. As shown in Fig. 1A, normal human thyroid tissues (lanes 1 and 5) have high levels of NICD protein (ratio of 1.0 when compared to GAPDH). Three primary thyroid cancers (2 PTC [lanes 2 and 3] and 1 FTC [lane 6] had much lower levels of NICD. Interestingly, a PTC tumor isolated from a lymph node metastasis had virtually no NICD protein (lane 4). These data illustrate that human thyroid cancers have minimal to no detected Notch1 protein whereas normal human thyroid tissue has abundant Notch1, supporting the idea that Notch may play a tumor suppressive role in thyroid cancer.
Similar to finding with human thyroid cancer specimens, we observed there was minimal amount of active Notch1 in PTC and FTC cells. To determine the role of Notch1, PTC DRO cells and FTC236 cells were transfected with the constitutive Notch1 plasmid and strong expression of Notch1 protein was induced as expected. Transfection of the Notch1 plasmid led to down-regulation of cyclin D1 and up-regulation of p21 (Figs. 1B and 1C). In addition, WI-38 fibroblast cells were also transfected with the NICD plasmid. Although high levels of Notch1 were achieved, there was no change in cyclin D1 and p21 levels (Fig. 1D). To determine how the cell growth was impacted by the NICD plasmid transfection and subsequent alteration of cyclin D1 and p21, we performed MTT assays. Overexpression of Notch1 in thyroid cancer cells FTC236 (Fig. 2A) resulted in a significant reduction in cellular proliferation whereas DRO showed statistically significant marginal reduction with (Fig. 2B). Importantly, Notch1 overexpression had no effect on the growth of WI-38 (Fig. 2B), suggesting that the growth suppressive effects of Notch1 are cell-type specific.
Based on our recent reports describing the ability of HDAC inhibitors to increase Notch1 protein levels in NET cells (17–20), we hypothesized that treatment of thyroid cancer cells with VPA and SBHA would also activate the Notch1 pathway. To assess the effects of VPA and SBHA on Notch1 protein expression, we performed Western blot analysis. As shown in Fig. 3A and 3B, both VPA and SBHA led to an increase of Notch1 in thyroid cancer cells. The levels of Notch1 increased with greater concentrations of VPA and SBHA in a dose dependent manner. Interestingly, even at very low concentrations, VPA (1 mM) and SBHA (10 µM) treatments led to increases in cleaved Notch1 protein. Consistent with their activity to induce Notch1 expression, VPA and SBHA inhibited HDAC and led to increased expression of AH4 in a dose-dependent manner (Fig. 3A and 3B). While treatment with HDAC inhibitors resulted in an increase in the cleaved form of Notch1 by Western blot, we wanted to determine if this protein was functional. Thus, we carried out a standard CBF-1 binding luciferase reporter assay. Active Notch1 binds with CBF-1 and other proteins to form a DNA-binding complex. This complex activates transcription of target genes. As shown in Fig. 3C, 2 mM and 4 mM VPA treatment of thyroid cancer cells transfected with the luciferase construct resulted in a two-fold and four-fold increase in relative luciferase activity, respectively, while 20 µM SBHA treatment of thyroid cancer cells resulted in a six-fold increase over control, indicating that the increase in CBF-1 binding was a result of induction of active, cleaved Notch1 (NICD). This result is consistent with the amount of Notch1 produced by HDAC inhibitors treatment.
HDAC inhibitor treatment has been shown to inhibit the growth of a variety of human cancer cells, including multiple myeloma (22), lymphoid cancers (23), malignant glioma (24), medulloblastoma (25,26), neuroblastoma (27), endometrial cancer (28), cervical cancer (29), ovarian cancer (30). Our previous reports showed that induction of Notch1 by HDAC inhibitors contributed to the cell growth inhibition in medullary thyroid cancer (19,20) and carcinoid cancer (17,18). We utilized the MTT assay to measure cell viability after VPA and SBHA treatment follicular-derived thyroid cancer cells. Thyroid cancer cells treated with VPA had a profound dose-dependent inhibition of growth (Fig. 4A). Statistically significant growth inhibition was also seen in thyroid cancer cells treated with SBHA (Fig. 4B) (p<0.05).
Next, we performed RNA interference assays as described to determine if HDAC inhibitors mediated growth inhibition is dependent upon Notch1 induction. Thyroid cancer cells were transiently transfected with siRNA against Notch1 or nonspecific siRNA. In the presence of nonspecific siRNA, VPA and SBHA treatment led to cellular growth inhibition as demonstrated above. However Notch1 siRNA blocked the antiproliferative effect of VPA and SBHA (Fig. 4C and D). These data strongly support the antiproliferative effect of Notch1 on thyroid cancer cells, and suggest that the growth inhibition seen with VPA and SBHA treatment were mediated by Notch1 signaling.
After establishing that Notch1 activation inhibits cellular proliferation in thyroid cancer cells, we were interested in determining the mechanism of action for this effect. We performed Western blot analysis after 2 days treatment with VPA to measure the effect of the drug on cell cycle-related genes expression. Treatment of thyroid cancer cells with VPA resulted in an increase in protein levels of the cyclin-dependent kinase inhibitors p21, p15 and p27 (Fig. 5A). The cell cycle promoter cyclin D1 was also suppressed by VPA.
We next performed siRNA interference assay to determine the effect of Notch1 blockage on the expressions of cell cycle-related genes. In the presence of nonspecific siRNA, VPA and SBHA treatment led to Notch1 expression as previously demonstrated (Fig. 5B, lane 2, 3). Notch1 siRNA completely blocked Notch1 induction by VPA or SBHA (Fig. 5B lanes 5, 6). The down-regulation of cyclin D1 and up-regulation of p21 (Fig 4B lane 2, 3) induced by VPA and SBHA were also blocked in cells transfected with Notch1 siRNA (Fig. 5B lanes 5, 6). Again, these data strongly indicated the cell cycle arrest effects by HDAC inhibitors were mediated by Notch1 signaling.
Follicular-derived thyroid cancers are usually sensitive to conventional therapy. The prognosis of most patients with differentiated thyroid cancer is very good (6,7), and most patients survive long disease-free intervals after appropriate thyroid surgery, and when necessary, radioiodine I131 therapy (8). Unfortunately this is not true of 30% of the cases, which develop toward dedifferentiation. The dedifferentiation process can lead to poorly differentiated thyroid cancer cells, which undergo a reduction in or disappearance of thyroid stimulating hormone (TSH) receptor and thyroglobulin expression, and therefore lose their ability to capture iodine. Lack of radioiodine uptake in thyroid cancer is usually associated with increased growth rate and larger tumor load and is seen in approximately 50% of patients with distant metastases (31). The ability to capture iodine is clinically important since it is associated with to the tumor’s sensitivity to radioiodine therapy. In many patients with identifiable I131 -negative lesions, surgery and radiotherapy may not be feasible owing to inoperability, previous radiotherapy, or additional distant metastases. Meanwhile, conventional chemotherapy is often ineffective. Thus, alternative treatment options are needed.
Notch1 is a multifunctional transmembrane receptor that regulates cellular differentiation, development, proliferation, and survival in a variety of contexts. However, the role of Notch1 in cancer cells remains controversial. Recent studies demonstrate that Notch1 signaling can act as either a tumor suppressor or as a tumor promoter, transient expression of active Notch1 in small cell lung cancer, pancreatic carcinoid, and prostate cancer cells inhibits cell growth in vitro ( see review in reference (12)). We have shown that stable expression of Notch1 (NICD) in pancreatic carcinoid BON and TT cells (16,32) leads to growth inhibition and reduction in neuroendocrine hormone production. However, the role of Notch1 signaling in follicular-derived thyroid cancer cells has, until now, not been described.
In this study, we have shown that active Notch1 (NICD) is minimal in thyroid cancer cells. NICD induction by VPA and SBHA led to dose responsive increase in functional Notch1 protein production as measured by western blot and CBF1 binding studies, resulting in activation of the Notch1 pathway. Furthermore, continuous Notch1 activation in thyroid cancer cells inhibited tumor cell growth. Notably, this growth reduction was dependent on the levels of Notch1 protein present. RNA interference experiments confirmed that these effects of VPA and SBHA on cell proliferation are mediated by Notch1 signaling. Our findings provide the first documentation of the role of Notch1 signaling as a tumor suppressor in thyroid cancer cells.
Moreover, the findings of this study suggest that the strong inhibition of thyroid cancer cells growth by Notch1 activation may be due to alterations in cell cycle related gene expression. p21 is a universal inhibitor of cyclin-dependent kinases. The observations of up-regulation of p21 and down-regulation of cyclin D1 following the activation of Notch1 suggest that growth inhibition of FTC cells may be due to cell cycle arrest.
Histone deacetylase (HDAC) inhibitors comprise a diverse group of structurally heterogeneous compound that exert antineoplastic effects in a variety of cancers in vitro and in vivo including multiple myeloma, lymphoid cancer, malignant glioma, neuroblastoma, cervical and ovarian cancer, and melanoma. VPA or SBHA has been shown, by our group, to inhibit a variety of NETs, including medullary thyroid cancer (19,20), gastrointestinal and lung carcinoid cancer (17,18) through the induction of Notch1. To our knowledge, this paper is the first report indicating the activation of Notch1 by HDAC inhibitors and cell growth suppression in follicular-derived thyroid cancer cells. Based on these findings, a clinical trial will be initiated at our institution to evaluate the effectiveness of activating Notch1 signaling by HDAC inhibitors for the treatment of patients with advanced and poorly differentiated thyroid cancer.
Grant Support: American Cancer Society Research Scholars Grant 05–08301TBE; National Institutes of Health Grants CA117117 and CA109053; American College of Surgeons George H.A. Clowes Jr. Memorial Research Career Development Award; Carcinoid Cancer Foundation Research Grant and the Society of Surgical Oncology Clinical Investigator Award.