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Medullary thyroid cancer (MTC) is a relatively uncommon neuroendocrine tumor that arises from the calcitonin-secreting parafollicular cells of the thyroid gland. Unfortunately, MTC frequently metastasizes, precluding curative surgical resection and causing significant morbidity. Thus, there is an urgent need for new treatment modalities. Tautomycin (TTY) and tautomycetin (TMC) are antifungal antibiotics isolated from Streptomyces spiroverticillatus and Streptomyces griseochromogens, respectively. GSK-3ß is a serine/threonine protein kinase that regulates multiple cellular processes and has been shown to be important in various cancers, including MTC. Treatment with TTY and TMC decreased neuroendocrine markers, suppressed hormonal secretion, and inhibited growth through apoptosis in MTC cells. Importantly, we describe a novel action of these compounds: inhibition of GSK-3ß.
Medullary thyroid cancer (MTC) is a neuroendocrine (NE) tumor that arises from the calcitonin-secreting parafollicular cells of the thyroid gland. MTC is relatively uncommon, representing only 3% of all thyroid malignancies; nevertheless, it accounts for 14% of all thyroid cancer-related deaths (1, 2). Unfortunately, MTC is difficult to treat, metastasizing in more than 50% of cases, which precludes surgical resection and causes significant morbidity (2). Surgery is the only curative therapy for MTC, and chemotherapeutic regimens are ineffective. Thus, there is an urgent need for new, targeted treatment modalities.
Glycogen synthase kinase-3ß (GSK-3ß) is a serine/threonine protein kinase that regulates multiple cellular processes including differentiation, metabolism, proliferation, and survival through targets such as ß-catenin, c-myc, and c-Jun (3). GSK-3ß is highly active and non-phosphorylated in unstimulated cells, and its activity is inhibited by phosphorylation of a single serine residue at position 9 (Ser9). There is a wide range of pharmacologic inhibitors of GSK-3ß, such as lithium, SB216763, and SB415286 (4). With such diverse effects, the GSK-3ß pathway has been shown to limit growth of various cancers, including pancreatic (5), prostate (6), and MTC (7).
The antifungal antibiotics tautomycin (TTY) and tautomycetin (TMC) are isolated from Streptomyces spiroverticillatus and Streptomyces griseochromogens, respectively (8, 9). These two compounds are structurally similar, differing only in the presence of a spiroketal group on TTY. In addition, TTY and TMC are potent inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A), a family of dephosphorylating enzymes (10). TMC was recently shown to inhibit the growth of colorectal cancer cells (11). However, the role of TTY and TMC in other cancers remains unclear. We present here the utilization of TTY and TMC for in vitro treatment of MTC. TTY and TMC decreased NE markers, suppressed hormonal secretion, and inhibited growth through apoptosis. Moreover, these effects were mediated through inhibition of GSK-3ß, a novel action of these compounds.
Human MTC cells (TT) were obtained from American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 medium (Life Technologies, Rockville, MD) supplemented with 18% fetal bovineserum (Sigma-Aldrich, St Louis, MO), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Life Technologies) in a humidified atmosphere of 5% CO2 in air at 37°C as previously described (7, 12).
To perform the MTT growth assay with TTY and TMC, 100,000 MTC or 20,000 NIH 3T3 (murine fibroblast) cells were seeded in quadruplicate on 24-well plates and incubated overnight. After incubation, MTC cells were treated with DMSO as control and increasing concentrations of TTY and TMC. Cells were incubated for up to 6 days. Every 2 days, treatment media was changed, and the MTT assay was performed by replacing the treatment medium with 250 μL of serum-free RPMI 1640 medium containing MTT (0.5 mg/mL) and incubating at 37°C for 4 hours. Next, 750 μL DMSO 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).
TTY and TMC were synthesized and dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich). MTC cells were treated with concentrations of TTY and TMC of up to 1000 nM, and an equal volume of DMSO was used as a control. After two days of treatment, whole cell lysates were prepared as previously described (13). Total protein concentrations were quantified with a bicinchoninic acid assay kit (Pierce Biotechnology, Rockford, IL). Denatured cellular extracts were resolved by SDS-PAGE, transferred onto nitrocellulose membranes (Schleicher and Schuell, Keene, NH), blocked in milk, and incubated with appropriate antibodies.
The antibody dilutions were as follows: 1:500 for chromogranin A (CgA; Zymed Laboratories, San Francisco, CA); 1:1,000 for achaete scute complex-like 1 (ASCL1; BD Biosciences, San Diego, CA), phosphorylated GSK-3ß (pGSK-3ß), GSK-3ß, phosphorylated PP1, PP1, Akt phosphorylated at serine 473 (pAkt), total Akt, poly-ADP ribose polymerase (PARP), cleaved caspase-3, and ß-actin (Cell Signaling Technology, Beverly, MA); and 1:10,000 for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Trevigen, Gaithersburg, MD).
Horseradish peroxidase conjugated goat anti-rabbit IgG (1:2000, Cell Signaling Technology) secondary antibody was used for CgA, pGSK-3ß, GSK-3ß, phosphorylated PP1, PP1, pAkt, total Akt, PARP, cleaved caspase-3, and GAPDH, while goat anti-mouse IgG (1:200, Pierce Biotechnology) secondary antibody was used for ASCL1. For visualization of the protein signal, SuperSignal West Femto (Pierce Biotechnology) was used for ASCL1 and cleaved caspase-3. Immunstar (Bio-Rad Laboratories, Hercules, CA) was used for CgA, pGSK-3ß, GSK-3ß, PARP, and GAPDH per the manufacturer's instructions.
To measure cellular effects after gene knockdown, MTC cells were plated onto 6-well plates and allowed to adhere overnight. The next day, cells were treated with lipofectamine (Invitrogen), 75 nM non-specific siRNA (Santa Cruz Biotechnology, Santa Cruz, CA), and 75 nM GSK-3ß siRNA (Santa Cruz Biotechnology) according to the manufacturer's instructions. After 2 days, cells were harvested and lysed as described above and analyzed for expression of ASCL1, GSK-3ß, and CgA. Relative calcitonin levels were measured as described below.
An enzyme-linked immunosorbent assay (ELISA) kit (Invitrogen) was utilized to quantify the amount of calcitonin in MTC cells as previously described (7, 14). MTC cells were treated with increasing concentrations of both TTY and TMC for 48 hours. Cell lysates were then used as the antigen source in a standard sandwich ELISA per the manufacturer's instructions. Samples were analyzed in triplicate.
Apoptosis was measured by the quantitation of cytosolic mono- and oligonucleosome-bound fragmented DNA by using an ELISA kit (Roche Applied Biosciences, Indianapolis, IN) as previously described (14). Briefly, MTC cells were treated with TTY and TMC (up to 1000 nM). After 48 hours, cell lysates were harvested and the cytosolic fraction was prepared. The lysate was used as an antigen source in an ELISA that consisted of a primary anti-histone antibody and a secondary anti-DNA antibody coupled to peroxidase. Absorbance values were used to calculate the induction of DNA fragmentation in comparison to control, and all samples were measured in quadruplicate.
One-way analysis of variance (ANOVA) and the independent samples T test were performed using SPSS (Version 11; SPSS, Inc., Chicago, IL) as appropriate to presented data. A P value of ≤ 0.05 was considered to be significant. All points represent the average readings plus/minus standard error.
To begin, we first wanted to see if TTY and TMC could affect cellular proliferation. We utilized an MTT growth assay to determine the impact of TTY and TMC on MTC cell growth. Growth was inhibited in a dose-dependent manner by both compounds in doses of up to 1000 nM (Figure 1a and 1b). Importantly, growth was suppressed significantly (18% by TTY and 10% by TMC, P < 0.01, one-way ANOVA) after only 2 days of treatment. This was even more statistically significant (P < 0.001) after 4 days. Additionally, TTY and TMC did not significantly affect the growth of murine fibroblasts until higher doses were utilized (Figure 1c and 1d).
After observing that both TTY and TMC could inhibit the growth of MTC cells, we wanted to explore the effects of TTY and TMC on the NE markers ASCL1 and CgA (Figure 2a). Previously, we have shown that pharmacologic inhibition of GSK-3ß in MTC cells leads to growth inhibition and a decrease of the NE markers ASCL1 and CgA (7). ASCL1 is a basic helix-loop-helix transcription factor that promotes neuronal differentiation and is a protein marker for NE hormone production (15). CgA is an acidic glycoprotein cosecreted with hormones by MTC, and the reduction of CgA is correlated with decreases in hormonal secretion (16).
Treatment for 48 hours with up to 1000 nM of TTY and TMC led to a dose-dependent decrease in the NE markers ASCL1 and CgA. Previous results from our studies in MTC demonstrated that these decreases become more significant with both longer treatments and increased concentrations (7, 17). To confirm that TTY and TMC affect hormonal secretion, we carried out an ELISA for calcitonin, a secretory product of MTC cells (Figure 2b). TTY and TMC decreased calcitonin secretion in a dose-dependent manner, with a maximum decrease of 70% and 67% with 1000 nM TTY and TMC, respectively. This decrease was statistically significant (P < 0.001 for both, one-way ANOVA). Thus, TTY and TMC decrease NE markers and suppress hormonal secretion of MTC cells.
After observing that TTY and TMC affected both growth and NE markers in MTC cells, we wanted to understand the mechanism of action of these compounds. Given the known role of TTY and TMC as protein phosphatase inhibitors (18), we explored the effects of TTY and TMC on GSK-3ß. GSK-3ß, in contrast to other kinases, becomes inactivated by phosphorylation in response to signaling cascades. Moreover, GSK-3ß has been shown to be an important target in MTC cells: both nonspecific (lithium chloride) and specific (SB216763, and SB415286) GSK-3ß inhibitors lead to decreased hormonal secretion and inhibition of growth (7). Treatment for 48 hours with up to 1000 nM of TTY and TMC led to a dose-dependent phosphorylation of GSK-3ß, demonstrating inhibition of the protein (Figure 3).
To determine that these effects were due to direct inhibition of the GSK-3ß protein, we utilized siRNA against GSK-3ß in MTC cells. Treatment for 48 hours with 75 nM GSK-3ß siRNA caused a significant decrease in total GSK-3ß protein, ASCL1, and CgA compared to both control cells and cells transfected with 75 nM non-specific siRNA (Figure 4a). A calcitonin ELISA demonstrated a 48% decrease in GSK-3ß siRNA treated cells, demonstrating a statistically significant inhibition of hormonal secretion (P = 0.02, independent samples T test, Figure 4b). Thus, direct inhibition of GSK-3ß results in a decrease in NE markers, similar to the results obtained by pharmacologic inhibition of the protein by TTY and TMC.
As previously noted, TTY and TMC are known protein phosphatase inhibitors (18). As such, we explored the effects of TTY and TMC on PP1. Increasing doses of TMC led to an increase in phosphorylated PP1 with a concomitant decrease in total PP1 protein, while increasing doses of TTY only decreased the total amount of PP1. Both of these actions suggest an inhibition of PP1. Thus, the known action of TTY and TMC as inhibitors of PP1 also occurs in MTC cells, which leads to inactivation of the GSK-3ß pathway (Figure 5a). Also, we measured the effects of TTY and TMC on a known upstream protein of GSK-3ß, Akt. Treatment with both TTY and TMC had no effect on phosphorylation of Akt at the serine 473 sites and total Akt (Figure 5b). In total, these data suggest that TTY and TMC act in a similar way as described in other cell lines by inhibiting PP1, which then inhibits GSK-3ß.
We wanted to explore the mechanism of growth inhibition. After 48 hours of treatment with TTY and TMC, cellular lysates were prepared. A Western blot was carried out for PARP and caspase-3, two well-characterized markers of the apoptotic pathway whose cleavage is indicative of apoptosis. After 48 hours of treatment, a dose-dependent cleavage was observed in both PARP and caspase-3 (Figure 6a).
To confirm that the compounds caused a significant amount of apoptosis, a DNA-fragmentation cell death ELISA was performed. This demonstrated a dose-dependent increase in fragmented DNA for both TTY and TMC (Figure 6b). There was an approximately 13-fold increase in DNA fragmentation at the highest doses of TTY and TMC used. These results indicate that TTY and TMC cause growth inhibition via apoptosis in MTC cells.
MTC is a rare tumor that accounts for 3% of thyroid malignancies and 14% of thyroid cancer deaths (1, 2, 19). Derived from the parafollicular calcitonin-secreting cells of the thyroid gland, these NE tumors frequently metastasize and cause a poor quality of life. Unfortunately, there are limited therapeutic options for patients with these tumors. As surgical resection is the only potentially curative treatment, there is an urgent need for new treatment modalities. We present here the utilization of two antifungal compounds, TTY and TMC, and their novel role as inhibitors of GSK-3ß in MTC.
TTY and TMC are naturally occurring antifungal compounds. TMC is isolated from Streptomyces griseochromogens, while TTY is isolated from Streptomyces spiroverticillatus, respectively (8, 9). Both TTY and TMC are potent inhibitors of PP1 and PP2A (10). Additionally, TMC inhibits the growth of colorectal cancer cells, demonstrating that these agents may be useful as anticancer drugs (11). Having demonstrated that pharmacologic inhibition of GSK-3ß has antiproliferative effects in MTC (7), we wanted to know if TTY and TMC may be able to inhibit GSK-3ß, limit NE markers, and suppress hormonal production of MTC cells.
In this study, we demonstrate conclusively that GSK-3ß plays a key regulatory role in cellular proliferation and NE markers in MTC cells. Gene knockdown experiments with siRNA for GSK-3ß led to a decrease in the NE markers ASCL1 and CgA. We have previously shown that these proteins are markers for decreased hormonal secretion, which does not appear to be due to cell death (7, 20). Transfection of GSK-3ß siRNA also caused significant hormonal suppression, an important component of targeted therapies for NE tumors. While we have previously demonstrated pharmacologic inhibition of GSK-3ß in MTC (7), we now show the key role that the protein plays via specific gene knockdown experiments, emphasizing the role of GSK-3ß as a therapeutic target in MTC.
Most importantly, we demonstrate that TTY and TMC are novel, naturally occurring inhibitors of GSK-3ß in MTC. Compared to other inhibitors used in MTC cells, TTY and TMC are significantly more potent than lithium chloride (millimolar) and of similar potency to SB216763 (micromolar) ranges. Moreover, the inhibition caused by TTY and TMC significantly decreases the NE markers ASCL1 and CgA and secretion of the NE hormone calcitonin. These effects appear to be due to direct GSK-3ß inhibition through inhibition of PP1, rather than less specific effects of TTY and TMC. These drugs also significantly inhibited growth of MTC cells via apoptosis. These data conclusively demonstrate that TTY and TMC inhibit GSK-3ß, affect NE markers, decrease hormonal production, and inhibit growth of MTC cells. Moreover, the growth effects of these compounds are not as pronounced in murine fibroblast cells, suggesting the specificity of these treatments for cancerous cells.
Overall, the present study is consistent with our earlier results in the targeting of GSK-3ß in MTC and pheochromocytoma cells (21). MTC, similar to other NE tumors, appears to have a wide range of molecular targets that regulate the NE phenotype and hormonal secretion, including Notch1, PI3K/Akt, and raf-1 (22). These results suggest that targeted molecular therapy of MTC is possible. Clearly, both TTY and TMC are molecular inhibitors of GSK-3ß in MTC cells.
In conclusion, TTY and TMC are novel, naturally occurring inhibitors of GSK-3ß in MTC cells. Treatment with TTY and TMC led to a decrease in NE markers and suppression of calcitonin secretion. Most importantly, TTY and TMC also led to growth inhibition via apoptosis in vitro. Crucially, this study further extends our understanding of the importance of GSK-3ß in MTC cells. We demonstrate here the utilization of two naturally occurring, potent compounds that inhibit GSK-3ß and may serve as viable compounds for future therapeutic use.
Grant support: Joel T. Adler and Mackenzie Cook are Howard Hughes Medical Institute Research Training Fellows. Additional support from the University of Wisconsin General Clinical Research Center (to J.T.A), National Institutes of Health Grant (CA113297 (to B.S.), a Research Scholars Grant from the American Cancer Society, National Institutes of Health Grants CA117117, and CA109053, the George H. A. Clowes, Jr., Memorial Research Career Development Award of the American College of Surgeons, a Carcinoid Cancer Foundation research award, the Clinical Investigator's Award from the Society of Surgical Oncology (to H.C.), and by a Carcinoid Cancer Foundation Research Award (to M. K.).